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CA 02872385 2014-11-27
COMPOSITIONS AND METHODS FOR GENETIC ANALYSIS OF POLYCYSTIC
KIDNEY DISEASE
Field of the Invention
=
The invention relates to a genetic testing method for identifying alterations
or the absence
of such alterations in a gene associated with Autosomal Dominant Polycystic
Kidney Disease.
Background of the Invention
Autosomal dominant polycystic kidney disease (ADPKD) is an exceptionally
common
hereditary nephropathology with an incidence of about 1 in 800 live births.
The disease is
progressive, phenotypically characterized by bilaterally enlarged polycystic
kidneys, and
typically resulting in end-stage renal disease (ESRD) by the age of 65 years.
The more common
complications include hypertension, macrohaematuria, urinary-tract infection,
cardiac-valve
abnormalities, and hernia of the anterior abdominal wall. Cyst formation is
also commonly
observed in the liver, although the occurrence is not associated with
functional impairment of the
organ. Although not as frequently reported, additional extrarenal
manifestations include
pancreatic cysts, connective tissue abnormalities, and cerebral-artery
aneurysms.
The typical age of onset is in middle life, but the range is from infancy to
80 years. The
clinical presentation of ADPKD differs between and within families as partly
explained by the
CA 02872385 2014-11-27
genetically heterogeneous nature of the disorder. Mutations in two genes, PICD-
1 and PKD-2,
account for nearly all cases of ADPKD (e.g., for reviews, see Arnaout, 2001,
Annu Rev. Med.
52:93-123; Koptides and Deltas, 2000, Hum. Genet. 107:115-126). PKD-1 and PKD-
2 encode
integral membrane proteins whose functions have not been fully elucidated. The
major gene
responsible for ADPKD, PKD-1, has been fully characterized and shown to encode
an integral
membrane protein, polycystin 1, which is thought to be involved in cell-cell
and cell-matrix
interaction. PKD-2 gene encodes polycystin-2 which is a predicted integral
membrane protein
with non-selective cation channel activity. Based on sequence homology with
the alpha 1
subunit component of voltage-activated calcium channels, it has been
postulated that polycystin-
2 may play a role in ion channeling. The C-terminal cytoplasmic tails of
polycystin-1 and
polycystin-2 have been shown to interact using in vitro binding assays and in
a directed two-
hybrid interaction. The interaction occurs via a coiled-coil domain in PICD-1
and a region near
R872 in PKD-2. Although the biological relevance of the interaction between
the polycystins is
not yet understood, it does suggest that PKD-1 and P1CD-2 are likely to
function along a common
pathway.
Both ADPKD type 1 and type 2 share the entire range of renal and extrarenal
manifestations, but type 2 appears to have a delayed onset relative to type 1.
The common
phenotypic complications observed for ADPKD including hypertension, hematuria,
and urinary
tract infection seem to be clinically milder in type 2 patients. The median
age at death or onset
of ESRD has been reported as 53 years in individuals with PKD-1 and 69 years
in those with
PKD-2. Women have been reported to have a significantly longer median survival
of 71 years
than men (67 years). No sex influence is apparent in P1CD-1. Mutations in the
P1CD-1 gene are
the cause of ADPKD in approximately 85% of the cases tested, while those in
PKD-2 account
for 15%. Although a small subset of families with ADPKD fail to demonstrate
genetic linkage
to either PKD-1 or P1CD-2, raising the possibility of a third gene for ADPKD,
the existence of a.
third disease-associated locus has been strongly challenged.
Despite the discovery of strong links between genetic alterations in PKD genes
and the
onset of ADPKD, the development of a genetic testing method for ADPKD
predisposition for
routine clinical use has been hindered by several technical obstacles.
2
CA 02872385 2014-11-27
One serious obstacle for developing a DNA-based testing method for ADPKD is
that
sequences related to the PKD transcript, for example, PKD-1, are duplicated at
least three times
on chromosome 16 proximal to the PKD-1 locus, forming PKD-1 homologues.
Another obstacle
is that the PKD-1 genomic interval also contains repeat elements that are
present in other
.. genomic regions. In addition, the sequences of PKD genes are extremely GC
rich and a large
number (15,816 bp) of nucleotides need to be analyzed for a thorough
evaluation.
There is a need for the identification of segments of these sequences that are
unique to the
expressed PKD genes and not are present in the duplicated homologous
sequences. There is also
a need for developing a sensitive and specific genetic testing method for
mutational analysis of
PKD genes. The development of such genetic testing method would facilitate the
diagnosis and
management of ADPKD.
Summary of the Invention
In one aspect, the present invention provides a method of mutation analysis of
a target
nucleic acid, the method comprising: incubating a sample comprising the target
nucleic acid in a
reaction mixture, in the presence of at least one first nucleic acid and at
least one second nucleic
acid, where the first nucleic acid comprises a primer sequence which anneals
to a unique site of a
sequence of SEQ ID NO. 1 or 2, and the second nucleic acid has an opposite
orientation from the
first nucleic acid, and where the incubation produces amplified products;
generating duplexes in
the amplified products; and detecting the presence or absence of a
heteroduplex from the
duplexes, where the presence of a heteroduplex indicates the presence of a
potential mutation in
the target nucleic acid, and where the absence of a heteroduplex indicates the
absence of a
mutation in the target nucleic acid.
In one embodiment, the method further comprises determining the sequence of a
heteroduplex region; and comparing the sequence of the heteroduplex region to
SEQ ID
NO. I or 2; where a sequence difference in the heteroduplex region compared to
SEQ ID
NO. 1 or 2 resulting in a predicted functional change in the protein encoded
by the target
nucleic acid is indicative of a mutation in the target nucleic acid.
3
CA 02872385 2014-11-27
Preferably, the first or second nucleic acid comprises a sequence selected
from the group
consisting of SEQ ID NOs. 3-49.
In another embodiment, the method further comprising performing a nested
amplification
reaction using the amplified products generated by the first and second
nucleic acids as templates
and generating duplexes in amplified products from the nested amplification.
Preferably, the nested amplification reaction is performed using at least one
primer
selected from the group consisting of SEQ ID NOs. 3-49 and their complementary
sequences.
In a preferred embodiment, the presence or absence of a heteroduplex from the
= duplexes is identified by DHPLC.
In also a preferred embodiment, the sequence of the heteroduplex region is
determined by
DNA sequencing.
Preferably, the second nucleic acid of the subject method comprises a primer
sequence
which anneals to a unique site within a sequence of SEQ ID NO. 1 or 2.
Also preferably, the sample comprising the target template is selected from
the group
consisting of: genomic DNA, cDNA, total RNA, mRNA, and a cell sample.
In one embodiment, the incubating step comprises an amplification reaction
selected from the group consisting of: a polymerase chain reaction, a ligase
chain reaction
(LCR) and a nucleic acid-specific based amplification.
The subject method of the invention may further comprise confirming the
amplified
product is a PKD-specific product with one or more restriction enzymes.
Preferably, the restriction enzyme cleaves a PKD-specific product to generate
a digestion
pattern distinguishable from a PKD homologue product.
More preferably, the restriction enzyme is selected from the group consisting
of: Pst I,
Stu I, Xma I, Mlu I, Pvu II, BssHII, Fsp I, Msc I, and Bln I.
4
CA 02872385 2014-11-27
In another aspect, the invention provides a diagnosis method for identifying a
patient affected with PKD, the method comprising:
(a) obtaining a sample from an individual;
(b) incubating the sample in a reaction mixture, in the presence of at
least one first
nucleic acid and at least one second nucleic acid, where the first nucleic
acid comprises a primer
sequence which anneals to a unique site within a sequence of SEQ ID NO. 1 or
2, and the second
nucleic acid has an opposite orientation from the first nucleic acid, and
where the incubation
produces amplified products;
(c) generating duplexes in the amplified products;
(d) detecting the presence or absence of a heteroduplex from the duplexes,
and(e)
determining the sequence of the heteroduplex region where the presence of a
mutation in
the heteroduplex region as compared to SEQ ID No. 1 or 2 is indicative that
the individual is
=
affected with PKD.
=
Preferably, the detection of a hereroduplex is performed by DHPLC.
Also preferably, the sequence is determined by DNA sequencing.
In one embodiment, the second nucleic acid comprises a primer sequence which
anneals
to a unique site within a sequence of SEQ ID NO. 1 or 2.
In another embodiment, the first or second nucleic acid comprises a primer
sequence
selected from the group consisting of SEQ ID NOs. 3-49.
The diagnosis method of the invention may further comprise performing a nested
amplification reaction using the amplified products generated by the first and
second nucleic
acids as templates and generating duplexes from the nested amplification.
In one embodiment, the nested amplification reaction is performed using at
least one
primer selected from the group consisting of SEQ ID NOs. 3-49 and their
complementary
, 25 sequences.
5
CA 02872385 2014-11-27
Preferably, the sample in the diagnosis method is selected from the group
consisting of: a
genomic DNA, cDNA, total RNA, mRNA, and a cell.
Also preferably, the amplification reaction is selected from the group
consisting
of: a polymerase chain reaction, a ligase chain reaction (LCR) and a nucleic
acid-specific
.. based amplification.
The diagnosis method may further comprise verifying the specifically amplified
product
with one or more restriction enzymes.
Preferably, the restriction enzyme cleaves a PKD-specific product to generate
a digestion
' .. pattern distinguishable from a PKD homologue product.
More preferably, the restriction enzyme is selected from the group consisting
of:
Pst I, Stu I, Xma I, Mlu I, Pvu II, BssHII, Fsp I, Msc I, and Bin I.
In a further aspect, the invention provides one or more nucleic acid primer,
where
each primer is an isolated nucleic acid selected from the group of SEQ ID NOs
3-49, or
the complement thereof.
The invention also provides a pair of nucleic acids, where at least one
nucleic acid
of the pair is selected from the group of SEQ ID NOs 3-49.
Preferably, the pair of nucleic acids have an opposite orientation and amplify
a fragment
of a template nucleic acid comprising a sequence of SEQ ID NO. 1 or 2.
In another aspect, the invention provides a composition comprising at least
one
isolated first nucleic acid and at least one isolated second nucleic acid,
where the first
nucleic acid is selected from the group of SEQ ID NOs. 3-49 and their
complementary
sequences, and the second nucleic acid has an opposite orientation from the
first nucleic
acid, and wherein the first and second nucleic acids amplify a fragment of a
template
nucleic acid comprising a sequence of SEQ ID NO. I or 2.
In one embodiment, the composition of the invention further comprises at least
one component selected from the group consisting of: a DNA poiymerase, a
template
6
CA 02872385 2014-11-27
nucleic acid, a restriction enzyme, one or more control oligonucleotide
primers, ddNTPs,
a PCR reaction buffer and their combination thereof.
Preferably, the template nucleic acid in the composition is a genomic DNA or
cDNA.
In a further aspect, the invention provides a kit for identifying a PKD
patient, the
kit comprising at least one isolated first nucleic acid and at least one
isolated second
nucleic acid, where the first nucleic acid is selected from the group of SEQ
ID NOs. 1-49
and their complementary sequences, and the second nucleic acid has an opposite
orientation from the first nucleic acid, and where the first and second
nucleic acids
amplify a fragment of a template nucleic acid comprising a sequence of SEQ ID
NO. 1 or
2, and packaging materials therefore.
In one embodiment, the kit of the invention further comprises at least one
component selected from the group consisting of: a DNA polyrnerase, a template
nucleic
acid, a restriction enzyme, a control oligonucleotide primer, ddNTPs, a PCR
reaction
buffer and the combination thereof.
Preferably, the template nucleic acid in the kit is a genomic DNA or cDNA
molecule.
The invention provides an isolated nucleic acid comprising a sequence selected
from the
group consisting of SEQ ID NOs. 3-49 and their complementary sequences
thereof.
The invention provides a nucleic acid biomarker for ADPKD comprising a PKD-1
or
PKD-2 nucleic acid sequence comprising one or more nucleotide alterations as
disclosed in
Figure 14.
In one embodiment, the at least one of the one or more nucleotide alterations
consists of a
novel nucleotide alterations as disclosed in Figure 14.
The invention also provides a nucleic acid biomarker for ADPKD comprising a
PK.D-1 or
PKD-2 nucleic acid sequence comprising one or more novel nucleotide
alterations as disclosed
in Figure 14.
7
CA 02872385 2014-11-27
89 The invention provides a polypeptide biomarker for ADPKD comprising a
PICD-1 or
PICD-2 polypeptide sequence comprising one or more amino acid alterations as
disclosed in
Figure 14.
In one embodiment, at least one the one or more amino acid alterations
consists of a
novel amino acid alteration as disclosed in Figure 14.
The invention provides a polypeptide biomarker for ADPKD comprising a PICD-1
or
PICD-2 polypeptide sequence comprising one or more novel amino acid
alterations as disclosed
in Figure 14.
The present invention further provides a method for diagnosing ADPKD in an
individual, comprising identifying nucleotide sequence of PKD-1 or PKD-2 gene
of the
individual, where the existence of one or more nucleotide sequence alterations
in the
nucleotide sequence of P1CD-1 or PKD-2 gene as disclosed in Figure 14 is
indicative of
ADPKD in the individual.
The present invention further provides a method for determining in an
individual the
presence or absence of a mutant P1CD gene, comprising the steps of
a) identifying the nucleotide sequence of a PKD-1 or PKD-2 gene of the
individual;
b) comparing the nucleotide sequence of step a) to the nucleotide sequence
alteration in the
nucleotide sequence of a PKD-1 or PKD-2 gene as disclosed in Figure 14; and
c) detecting the presence of one or more of the nucleotide sequence
alterations disclosed in
Figure 14; wherein the presence of at least one of the nucleotide sequence
alterations is
indicative of ADPKD in the individual; and wherein the absence of any of said
nucleotide
sequence alterations indicates the absence of a mutant PKD-1 and/or PKD-2
gene.
In one embodiment, the method for diagnosing ADPKD and/or the method for
determining the presence or absence of a mutant PKD gene further comprises
obtaining a
DNA sample from the individual for the identification of nucleotide sequence
of PKD-1
or PKD-2 gene.
8
CA 02872385 2014-11-27
Preferably the DNA sample obtained is a genomic DNA sample or a cDNA
sample.
In another embodiment, the method for diagnosing ADPKD and/or the method for
determining the presence or absence of a mutant PKD gene further comprises
amplifying
a portion of the PKD-1 or PKD-2 gene from the DNA sample before the
identification.
Preferably, the portion of the PKD-1 or PKD-2 gene is amplified by a
polymerase
chain reaction.
Also preferably, the nucleic acid sequence is identified by DNA sequencing.
More preferably, the DNA sequencing is performed using an isolated nucleic
acid
comprising a sequence selected from the group consisting of SEQ ID NOs. 3-49
and their
complementary sequences thereof.
In one embodiment, the at least one or more of the at least one or more
nucleotide
alterations consists of a novel nucleotide alterations as disclosed in Figure
14,
= Brief Description of the Drawines
The objects and features of the invention can be better understood with
reference to the
following detailed description and accompanying drawings.
Figure 1 is a figure showing the PICD I cDNA sequence (GenBank Accesion No.
L33243)
used in one embodiment of the invention. Exon and PCR product junctions are
depicted above
the nucleotide sequence. Amino acids are positioned under the center of each
codon.
Figure 2 is a figure showing the comparison of exon sequences of a PKD gene
and two
homologue sequences according to one embodiment. Restriction enzyme sites
which only
cleave in either PKD or homologue sequence are indicated.
Figure 3 is a graph showing PKD I exon 40 DHPLC patterns of 4 normal samples
and a
19 bp insertion (duplication) at nucleotide 11606, codon 3799 according to one
embodiment.
9
CA 02872385 2014-11-27
Figure 4 is a graph showing PI(D1 exon 40 sequences of the normal control and
a
sequence with a 19 bp insertion (duplication) at nucleotide 11606, codon 3799
according to one
embodiment.
Figure 5 is a graph showing PK.D1 exon 6 DHPLC patterns of an intron 5
probable
polymorphism (IVS5-9 (i->A) and a frameshift at nucleotide 1502 (insert (3) in
two related
patients according to one embodiment.
Figure 6 is a graph showing PICD1 exon 6 sequences of the normal control and a
sequence with intron 5 probable polymorphism (IVS5-9 G->A) according to one
embodiment.
Figure 7 is a graph showing PICD1 exon 18 DHPLC patterns of a frameshift at
nucleotide
7518, codon 2436 (insert C), and a common polymorphism C7652T according to one
embodiment. '
Figure 8 is a graph showing PKD1 exon 18 sequences of the normal control and
a.
sequence with frameshift at nucleotide 7518, codon 2436 (insert C) according
to one
embodiment.
Figure 9 is a graph showing an example of a software-predicted melt profile
and the need
for multiple temperatures to establish partial melting near the ends of an
exon according to one
embodiment.
Figure 10A is a chart showing patient DNA variant genotypes determined in one
embodiment of the invention.
Figure 10B is a table showing patient DNA variant genotypes determined in one
embodiment of the invention.
Figure 11 is a table summarizing DHPLC (WAVE) conditions used in some
embodiments of the invention.
Figure 12 is a table summarizing PCR conditions used in some embodiments of
the
invention.
CA 02872385 2014-11-27
=
Figure 13 is a schematic diagram showing patient specimen processing steps in
one
embodiment of the invention.
Figure 14 is a table showing non-limiting examples of novel and known
nucleotide and
amino acid alterations identified in PKD-1 and PKD-2 nucleotide and amino acid
sequences
from ADPKD patients according to one embodiment of the invention. Novel, as
used herein,
includes the unknown predicted disease causing (UPD) alterations disclosed in
bold. X refers to
Exon, WS refers to intervening sequence, KP refers to known polymorphism, UP
refers to
unknown polymorphism, and UAA refers to unknown amino acid change.
Figure 15 is a wild-type PKD-1 cDNA sequence according to one embodiment of
the
invention.
Figure 16 is a wild-type PKD-2 cDNA sequence according to one embodiment of
the
invention.
Detailed Description of the Invention
=
The subject invention is based on the identification of unique sites within a
PKD gene,
the design of PKD-specific primers and the DHPLC analysis of PCR products
amplified by using
these PKD-specific primers.
I. Definitions
As used herein, "ADPKD" refers to autosomal dominant polycystic kidney
disease.
ADPKD is an exceptionally common hereditary nephropathology and is
characterizcd by the
development of renal cysts and, ultimately, renal failure, and may
alternatively or in addition
involve cysts in other organs including liver and spleen, as well as
gastrointestinal,
cardiovascular, and musculoskeletal abnormalities.
The term "PKD gene" refers to a genomic DNA sequence which maps to chromosomal
= position 16p13.3 (i.e., PKD-1) or chromosomal position 4q21-23 (i.e., PKD-
2) and gives rise to a
messenger RNA molecule encoding a PKD protein. The PKD-1 and PKD-2 genes
comprise the
= sequences of SEQ ID NO. 1 and SEQ ID NO.2, respectively, which include
introns and putative
regulatory sequences. Like many other genes, PKD-1 and PKD-2 gene sequences,
when
11
CA 02872385 2014-11-27
compared among individuals, show sequence variations. Those genes having
polymorphisms
which are silent (i.e., with respect to gene expression or function of a gene
product) are "normal"
genes as defined herein.
A "normal" PKD gene (e.g., PKD-1 or PKD-2) is defined herein as a PKD gene
such as
described by SEQ ID NO. 1 or 2, respectively, and includes any gene having
silent
polymorphisms.
A "mutant" PKD gene is defined herein as a PKD gene (e.g., PKD-1 or PKD-2)
whose
sequence is modified by mutation comprising one or more substitutions
(transitions or
transversions), deletions (including loss of locus), insertions (including
duplications),
translocations, and/or other modifications relative to the normal PKD gene.
The mutation causes
detectable changes, in the expression or function of the PKD gene product, and
is causative for
ADPKD. The mutations may involve from one to as many as several thousand
nucleotides, and
result in one or more of a variety of changes in PKD gene expression (e.g.
decreased or increased
rates of expression) or expression of a defective RNA transcript or protein
product. Mutant PKD
genes encompass those genes whose presence in one or more copies in the genome
of a human
individual is associated with ADPKD.
As used herein, "biomarker" refers to a biological molecule, e.g., a nucleic
acid or
polypeptide or peptide etc... whose presence or concentration can be detected
and correlated
with a known condition, such as a disease state, for example polycystic kidney
disease, and in
particular, ADPKD.
A "nucleotide sequence alteration" or "nucleotide alteration" refers to a
nucleotide
sequence modification including one or more substitutions (transitions or
transversions),
deletions (including loss of locus), insertions (including duplications),
translocations, and/or
other modifications relative to the normal PKD gene.
An "amino acid alteration" refers to an amino acid modification including a
substitution,
a frameshift, a deletion, a truncation and an insertion, and/or other
modifications relative to the
normal PKD amino acid sequence.
12
CA 02872385 2014-11-27
The term "basepair mismatch" refers to any nucleic acid sequence which is not
complementary to the sequence of SEQ ID. NO. 1 or 2. Therefore, basepair
mismatch,
according to the present invention may be caused by gene alteration or
polymorphism of a
normal PKD gene; or by any modifications present in a mutant PKD gene.
"Basepair mismatch"
may be a single nucleotide basepair mismatch or it may include a nucleic acid
sequence of 2 or
more nucleotides (i.e., 3, or 4, or 5, or 10, or 20, or 100, or 500 more, or
up to 1000 nucleotides).
The presence or absence of a mismatch, as defined herein, is indicative of the
presence or
absence of a potential mutation in the target nucleic acid.
The term "authentic" is used herein to denote the genomic sequence of SEQ ID.
NO. 1 or
.. 2, as well as sequences derived therefrom, and serves to distinguish these
authentic sequences
from "PKD homologues" (see below).
A "PKD-1 homologue" is a sequence which is closely related to PKD-1, but which
does
not encode an expressed PKD-:1 gene product. Several examples of such
homologues that map
to chromosomal location 16p13.1 or 4q21-23 have been identified and sequenced.
A PKD-1
homologue may share more than 95% sequence identity to an authentic PKD gene.
As used herein, a "specifically amplified product" is a product amplified from
a fragment
within an authentic PICD gene (e.g., SEQ ID NO. I or 2), but not from a PKD
homologue. A
"non-specifically amplified product" is a product amplified from a PKD
homologue or other
sequences due to the annealing of nucleic acid primers to a template sequence
which is not
completely complementary during the amplification reaction.
As used herein, a "unique site" refers to a stretch of sequence of 10-50 base
pairs in
length within a PKD gene which comprises at least one nucleotide different
form a stretch of
sequence in a PKD homologue or other sequences. One exemplary unique site
comprises a
sequence of 5' AGO TCC AGO GCG ACT CGC TUG 3', or 5' CAG GGC CAC ACG CGC
TOG GCG 3', or their complement thereof.
As used herein, a "PKD-specific primer" refers to a nucleic acid sequence
which anneals
to a sequence within a PICD gene (including introns and exons) under specific
stringent
conditions. A PKD-specific primer, according to the invention, anneals to a
unique site present
13
CA 02872385 2014-11-27
in the authentic expressed PKD-1 gene or PKD-2 gene, and not to PKD homologues
or other
sequences under specific stringent conditions. A PKD-specific primer shares
more then 95%
(e.g., more than 96%, 96%, 97%, 98%, 99%, or up to 100%) sequence identity
with a unique site
within a PKD gene. A "PKD-specific primer" may be 10 to 60 nucleotides in
length, for
.. example, 18-52 nucleotides in length.
As used herein, the term "specific stringent condition" refers to an
amplification
condition which specifically allows the annealing of a PKD-specific primer to
a sequence within
a PICD gene. Under a "specific stringent condition", a PKD-specific primer
does not anneal to a
PKD homologue or other sequences. For example, one specific stringent
condition useful to the
10, invention comprises a Taq Precision buffer (TaqPlus Precision buffer,
Stratagene, La Jolla, Cat#
600210), a dNTP concentration of more than 50 nM, for example, 100 nM, 200 nM,
or 300 nM.
The annealing temperature in a specific stringent condition may be higher than
or less than or
equal to 5 C below the lowest primer annealing temperature (Tm), for example,
1 C, 2 C, 4 C,
5 C, or 10 C higher than Tm or 4 C, 3 C, 2 C, or 1 C below Tm.
"Amplification" of DNA as used herein refers to a reaction that serves to
increase the
concentration of a particular DNA sequence within a mixture of DNA sequences.
Amplification
may be carried out using polymerase chain reaction (PCR), ligase chain
reaction (LCR), nucleic
acid-specific based amplification (NSBA), or any other method known in the
art.
"RT-PCR" as used herein refers to coupled reverse transcription and polymerase
chain
reaction. This method of amplification uses an initial step in which a
specific oligonucleotide,
oligo dl, or a mixture of random primers is used to prime reverse
transcription of RNA into
single-stranded cDNA; this cDNA is then amplified using standard amplification
techniques e.g.
P CR.
A "template nucleic acid" or a "target nucleic acid" (e.g., a genomic DNA or a
cDNA), is
a normal (e.g., wild type) or a mutant nucleic acid that is or includes a
particular sequence (e.g. a
PICD-1 or PKD-2 gene sequence). It will be understood that additional
nucleotides may be
added to the 5' and/or 3' terminus of the disclosed sequence, as part of
routine recombinant DNA
manipulations. Furthermore, conservative DNA substitutions i.e. changes in the
sequence of the
14
CA 02872385 2014-11-27
protein-coding region that do not change the encoded amino acid sequence, also
may be
accommodated.
As used herein, "nucleic acid primer" refers to a DNA or RNA molecule capable
of
annealing to a nucleic acid template and providing a 3' end to produce an
extension product
which is complementary to the nucleic acid template. The nucleic acid template
is catalyzed to
produce a primer extension product which is complementary to the target
nucleic acid template.
The conditions for initiation and extension include the presence of four
different
deoxyribonucleoside triphosphates and a polymerization-inducing agent such as
DNA
polymerase or reverse transcriptase, in a suitable buffer ("buffer" includes
substituents which are
cofactors, or which affect pH, ionic strength, etc.) and at a suitable
temperature. The primer
according to the invention may be single or double stranded. The primer is
single-stranded for
maximum efficiency in amplification, and the primer and its complement form a
double-stranded
nucleic acid. But it may be double stranded. "Primers" useful in the present
invention are less
than or equal to 100 nucleotides in length, e.g., less than or equal to 90, or
80, or 70, or 60, or 50,
or 40, or 30, or 20, or 15, or equal to 10 nucleotides in length.
As used herein, the term "opposite orientation", when referring to primers,
means that
one primer comprises a nucleotide sequence complementary to the sense strand
of a target
nucleic acid template, and another primer comprises a nucleotide sequence
complementary to the
antisense strand of the same target nucleic acid template. Primers with an
opposite orientation
may generate a PCR amplified product from matched nucleic acid template to
which they
complement. Two primers with opposite orientation may be referred to as a
reverse primer and a
forward primer.
As used herein, the term "same orientation'', means that primers comprise
nucleotide
sequences complementary to the same strand of a target nucleic acid template.
Primers with
same orientation will not generate a PCR amplified product from matched
nucleic acid template
to which they complement.
Alternatively, primers of the present invention may be labeled with a
detectable label
such as a radioactive moiety, or a fluorescent label, or alternatively, the
amplification reaction
CA 02872385 2014-11-27
may incorporate labeled nucleotides into the reaction product. Thus, the
amplification reaction
product may be "detected" by "detecting" the fluorescent or radioactive label.
As used herein, a "nucleic acid" generally refers to any polyribonucleotide or
poly-
.
deoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or
DNA.
"Nucleic acids" include, without limitation, single- and double-stranded
nucleic acids. As used
herein, the term "nucleic acid(s)" also includes DNAs or RNAs as described
above, that contain
one or more modified bases. Thus, DNAs or RNAs with backbones modified for
stability or for
other reasons are "nucleic acids". The term "nucleic acids" as it is used
herein embraces such
chemically, enzymatically or metabolically modified forms of nucleic acids, as
well as the
chemical forms of DNA and RNA characteristic of viruses and cells, including
for example,
simple and complex cells.
As used herein, "isolated" or "purified" when used in reference to a nucleic
acid means
that a naturally occurring sequence has been removed from its normal cellular
(e.g.,
chromosomal) environment or is synthesized in a non-natural environment (e.g.,
artificially
synthesized). Thus, art "isolated" or "purified" sequence may be in a cell-
free solution or placed
in a different cellular environment. The term "purified" does not imply that
the sequence is the
only nucleotide present, but that it is essentially free (about 90-95%, up to
99-100% pure) of
non-nucleotide or nucleic acid material naturally associated with it, and thus
is distinguished
from isolated chromosomes.
As used herein, "genomic DNA" refers to chromosomal DNA, as opposed to
complementary DNA copied from an RNA transcript. "Genomic DNA", as used
herein, may be
all of the DNA present in a single cell, or may be a portion of the DNA in a
single cell.
As used herein, "complementary" refers to the ability of a single strand of a
nucleic acid
(or portion thereof) to hybridize to an anti-parallel nucleic acid strand (or
portion thereof) by
contiguous base-pairing between the nucleotides (that is not interrupted by
any unpaired
nucleotides) of the anti-parallel nucleic acid single strands, thereby forming
a double-stranded
nucleic acid between the complementary strands. A first nucleic acid is said
to be "completely
complementary" to a second nucleic acid strand if each and every nucleotide of
the first nucleic
acid forms base-pairing with nucleotides within the complementary region of
the second nucleic
16
CA 02872385 2014-11-27
acid. A first nucleic acid is not completely complementary to the second
nucleic acid if one
nucleotide in the first nucleic acid does not base pair with the corresponding
nucleotide in the
second nucleic acid.
As used herein, a "sample" refers to a biological material which is isolated
from its
natural environment and containing target nucleic acid, and may consist of
purified or isolated
nucleic acid, or may comprise a biological sample such as a tissue sample, a
biological fluid
sample, or a cell sample comprising target nucleic acid.
As used herein, a "double stranded DNA" is referred to as a "duplex". When the
base
sequence of one strand is entirely complementary to base sequence of the other
strand, the
duplex is called a "homoduplex". When a duplex contains at least one base pair
which is not
complementary, the duplex is called,a "heteroduplex". In the subject
invention, the formation of
a heteroduplex, when amplified products from a sample taken from an individual
are denatured
and re-annealed, indicates the presence of a potential mutant PK.D gene in
that individual.
As used herein, "DHPLC" refers to a separation process called "denaturing high
performance liquid chromatography" which has been used to detect sequence
variants by
separating a heteroduplex (resulting from the presence of a mutation) and a
homoduplex having
the same bp length. This separation is based on the fact that a heteroduplex
has a lower melting
temperature (Tin) than a homOduplex. DHPLC can separate heteroduplexes that
differ by as
little as one base pair under certain conditions. DHPLC can also be used to
separate duplexes
having different bp in length.
The "heteroduplex site separation temperature" or "midpoint temperature" or
"Tm" is
defined herein to mean, the temperature at which one or more base pairs
denature, i.e., separate,
at the site of base pair mismatch in a heteroduplex DNA fragment.
General Description of PKD Genes
The PKD-1 gene (e.g., genbank accession number L39891, SEQ ID NO. 1) spans
about
54 kb of genomic DNA on chromosome 16 (16p13.3) and contains a 12,906 bp
coding sequence
divided into 46 exons from which a 14 kb mRNA is transcribed (Mochizuki et
al., 1996, Science,
272:1339-1342; Hughes et al., 1995, Nature Genet..10:151-160). The protein
product of PKD-1,
17
CA 02872385 2014-11-27
Polycystin-1, is a 4303 amino acid protein with a predicted mass of 460 kDa.
Until recently,
analysis of the PKD-1 gene had not been amenable to genetic analysis largely
because of the
presence of at least three highly homologous copies of the gene that map
proximal to PKD-1
along chromosome 16 (16p13.1). Approximately 75% of the PKD-1 gene is
duplicated and
shares about 97% identity with its homologous copies. The reiterated region
encompasses a 50
kb (5') portion of the gene containing the first 33 exons. Only the most 3',
5.7 kb of the gene,
containing exons 34-46, is unique to PKD-1. Another notable feature of the PKD-
1 gene is a
polypyrimidine tract in intron 21 that is 2.5 kb long, the longest described
in the human genome.
The PKD-2 gene (e.g., genbank accession number AF004859 ¨ 004873, SEQ ID NO.
2) spans
68 kb of genomic DNA and is located on chromosome 4 (4q21-23) (Mochizuki et
al., 1996,
supra). PKD-2 contains 15 exons and encodes a 5.4 kb transcript from which a
968-amino acid
protein product of approximately 110 kDa is generated. Mutation analysis of
PKD-2 is to a great
extent easier than that of PKD-1 because PKD-2 is a single copy gene. See
Table 1 for a
summary of PKD genes and their protein products.
Table 1. PKD gene description
Gene Description PKD-1 PKD-2
Chromosome 16p13.3 4q21-23
Genomic length 54 kb 68 kb
Exons 46 15
Base pairs 12909 2904
Codons 4303 968
Protein Polycystin-1 Polycystin-2
Based on evidence supporting the occurrence of somatic mutations on the normal
allele, a
two-hit model similar to the pathogenesis of the many familial cancer
predisposition syndromes
18
CA 02872385 2014-11-27
has been proposed to explain the clinically focal manifestations of the
disease (Qian et al., 1996,
Cell, 87:979-987; Watnick et al., 1998Mol. Cell. 2:247-251). Briefly, the
model suggests that
ADP1CD is recessive at the cellular level and that a second somatic mutation
or "hit" in a
heterozygous P1CD defective background would result in the homozygous loss of
P1CD function
in the affected renal tubular epithelial cell. The loss of PICD function is
postulated to disrupt the
signaling mechanisms required for proper cell differentiation and in turn
leads to the abnormal
proliferation of the afflicted cell into cystic structures.
Direct sequencing of the PKD-1 gene has revealed the presence of polymorphism
in
normal individuals and a multitude of different sequence alterations in ADPKD
affected
individuals. Table 2 shows a sypnosis of the P1CD-1 sequence alterations
described in the
literature to date.
Table 2: Published PKD-1 sequence alterations including mutations and
polymorphisms*
Codon Nucleotide Nucleotide
Fragment number
Amino Acid Change Consequence
NumberNumber Change
Intron 1-Exon 5 3kb del
5 224 1 13del
frameshift
88 474 ' 2 GCG-GTG Ala-Val
92 487 2 GCG-GCA Ala-Ala
polymorphism
225 885 5A + 53 TCG-TAG Ser-X
termination
227 890 5A + 53 CAG-TAG Gln-X
termination
230 900 5A + 5B TGC-TTC Cys-Phe
324 1182 5B + 5C CGC-CTC Arg-Leu
341 1234 5C GCC-GCT Ala-Ala
polymorphism
19
CA 02872385 2014-11-27
373 1330 5C CTT-CTC Leu-Leu
polymorphism
403 1420 6 CAC-CAT His-His
polymorphism
7 CAG-CAA splice
acceptor skip exon 7
570 1921 8 CAC-CAT
' His-His polymorphism
9 ' CAG-CAT splice
acceptor skip exon 9
695 2296 10 C del---ccc-cc^g Pro-Pro
frameshift
695 2296 10 C ins=ccc-cc"c Pro-Pro
frameshift
705 2324 11A CAG-TAG Gln-X
termination
738 2425 11A CCC-CCG Pro-Pro
polymorphism
749 2457 11A TCA-TGA Ser-X
termination
845 2745 11B TTG-TCG Leu-Ser
898 2905 1113 + 11C GCA-GCC Ala-Ala
polymorphism
900 2911 11B + 11C CCG-CCA Pro-Pro
polymorphism
910 2941 11B -F 11C GAC-GAT Asp-Asp
polymorphism
967 3110 12 TGG-CGG Trp-Arg
991 3183 12 GTC-GGC ' Val-Val
polymorphism
13 AGC-TGC splice
acceptor skip exon 13
1003 3220 13 4bp del=agc-ag^g Ser-Arg
frameshif1
1021 3274 13 GGT-GGC Gly-Gly
polymorphism
CA 02872385 2014-11-27
1037 3322 13 CTA-CTG Leu-Leu
polymorphism '
1041 3336 13 del g------ggc-ecg Gly-Ala
frameshift
14 AGG-AAG splice acceptor skip exon 14
1092 3486 14 CAT-CAC His-His
polymorphism
1124 3583 15A GCC-GCT Ala-Ala
polymorphism
1125 3586 15A TCC-TCT Ser-Ser
polymorphism
1166 3707 15A + 15B GGC-AGC Gly-Ser probable
path.
, 1198 3804 15B 7bp del=agc-egg Ser-Arg
frameshift
1288 4075 15C + 15D CAC-CAT His-His
polymorphism
1289 4077 , 15C + 15D t del=stg-egc - Val-Gly
frameshift
_ 1309 4137 15D ct del=cct _ -c"ga
Pro-Arg frameshift -
. ,
1346 4249 15D ac del=aca-ac"a Thr-Thr frameshift
, 1360 4291 , 15D + 15E g del=gtg-gt"c Val-Val
frameshift
1399 4406 15E TGG-CGG Trp-Arg
1525 4784 15G g del=gtt-Atta Val-Leu
frameshift
1537 4820 15G GAG-TAG Glu-X termination
1545 4846 15G AAG-AAA Lys-Lys
polymorphism
1555 4876 15G+ 15H GCA-GCC Ala-Ala
polymorphism
1558 4885 15G + 15H ACG-ACA Thr-Thr _
polymorphism
,
,
21
CA 02872385 2014-11-27
1563 4898 15G + 15H t ins=aat-a^ta Asn-Ile
frameshift
1633 5109 151 t ins=gag-gatg Glu-Asp
frameshift
1653 5168 151 CAG-TAG Gln-X
termination
1672 5225 151+ 15J a del=agg-^ggg Arg-Gly
frameshift
1672 5225 151 + 151 ag del=agg-Aggg Arg-Gly
frameshift
1724 5383 151 ACC-ACT . Thr-Thr polymorphism
.-.--
1786 I 5566 151 + 15K ____ CCG-CTG Pro-Leu
1787 5579 151+ I5K CTG-TTG Leu-Leu
polymorphism
1826 5689 ' 15K TGG-TGA Trp-X
termination
1829 5696 15K CTG-TTG Leu-Leu
polymorphism
1858 5783 15K g del=gat-^atg Asp-Met
frameshift
-.
1874 5833 15K TGG-TGA Trp-X
termination
14del=ccatc-
1887 5870 15K Ile-Val
frameshift
ccAget
1921 5974 15L - CTG-CTA Leu-Leu
polymorphism
1922 5975 15L CAG-TAG Gln-X
termination
lbp ins=eac-
1938 6024 15L His-** frameshift
caA**
1949 6058 15L AGC-AGT ¨ Ser-Ser ¨
polymorphism'
..._
1956 6078 15L GTG-GAG Val-Glu
probable path.'
22
CA 02872385 2014-11-27
1960 6089 15L CAG-TAG Gln-X
termination
1992 6187 15L 4bp del=ttc-tt"
** frameshift
1995 ' 6195 15L CGC-CAC Arg-His
polymorphism
2039 6326 15M + 15L CAG-TAG Gln-X
termination
2075 6434 15M 28bp del
frameshift
__________________________________________________ _ ______________
2144 6642 15M 27bp del
frameshift
. r
2163 6698 15M CGA-TGA Arg-X
termination
,
_
______________________________________________________________________________
2192 6785 15M + 15N 7bp del=acc-Aget Thr-Ala
frameshift
' 15bp del=cgg-
in frame
2220 6868 15N Arg-Val
Agtg
deletion .
2222 6876 15N GCG-GTG Ala-Val
_
______________________________________________________________________________
.
2229 6898 15N TGC-TGA Cys-X
termination
2242 6937 15N ac delr----aca-ac"a
Thr-Thr frameshift
2243 6938 15N CAG-TAG Gln-X
termination
2250 ' 6960 15N - ACG-ATG Thr-Met
15 GGT-GGG - splice donor
. _
16 CAG-GAG splice acceptor
skip exon16
_
______________________________________________________________________________
2309 7138 16 GGC-GGT Gly-Gly
polymorphism
2113 7147 16 CrCG-GCA Ala-Ala
polymorphism
,
,
23
,
,
CA 02872385 2014-11-27
2323 7179 16 14bp del.----gte-gt^ Val-X
termination
__________________________________________ _ ______________
2329 7196 16 CGG-TGG Arg-Trp
_
2332 7205 16 7del=gct-Atgg Ala-Trp
frameshift
2334 7211 16 7ins=gtg-Agtg Val-Val
frameshift
2336 7219 16 TAC-TAA Tyr-X
termination
17 CAG-GAG splice acceptor skip
exon 17
2370 7321 17 TGT-TGA Cys-X
termination
237/ 7324 17 gt del=gtg-gt^c - Val-Val
frameshift
2378 7345 17 GTG-GTT Val-Val
polymorphism
,
2379 7347 17 TAC-TGC Tyr-Cys
2389 7376 17 TTG-CTG Leu-Leu
polymorphism
L
2392 7386 17 CGC-CCC Arg-Pro
2396 7397 17 1 lbp ins¨att-Attg Ile-Leu -
frameshift
_ _____________________________________________________________________
2402 7415 17 CGA-TGA Arg-X
termination
2408 - 7433 18 ' CGT-TGT Arg-Cys
probable path.
_ _____________________________________________________________________
2423 7479 18 TCC-TTC Ser-Phe
2430 7499 18 CGA-TGA Arg-X
termination
3bp ins¨gag-
2442 7535 18 Glu-Gly
probable path.
g^gcg
24
CA 02872385 2014-11-27
2471 7623 18 CCG-CTG Pro-Leu
2481 7652 18 CTG-TTG Leu-Leu
polymorphism
2495 7696 18 TGC-TGT Cys-Cys
polymorphism
2519 7767 19 CAG-CTG Gln-Leu
2548 7853 19 GAG-CAG Glu-Gin
polymorphism
2558 7883 19 CAG-TAG Gln-X termination
' 2570 7919 20 TTG-CTG Leu-Leu polymorphism
Gly del in
2579 7945 20 gge del----ggc-^age Gly-Ser
frame
2582 7956 20 ACG-ATG Thr-Met
polymorphism
2597 8002 20 CCA-CCG Pro-Pro
polymorphism
2604 8021 20 GAT-AAT Asp-Asn
polymorphism
2607 8030 20 5bp dek-cae-Acat His-His frameshift
Leu del in
2612 8046 20 gtt del¨tegtt-teg Ser-Ser
frame
2638 8124 21 CAC-CGC His-Arg
polymorphism
2639 8126 21 CGA-TGA Arg-X termination
20 ins--ega-
2639 8126 21 Arg_** fraMeshift
e****
2649 8157 21 ACT-ATT Thr-Ile
CA 02872385 2014-11-27
2650 8159 21 del ct=ctg-Aggt Leu-Gly
frameshift
_ _____________________________________________________________________
2658 8183 21 8bp del Val-X
termination
2674 8231 22 CCC-TCC Pro-Ser
polymorphism
2696 8298 22 CTC-CGC Leu-Arg
2708 8334 22 ACG-ATG Thr-Met
polymorphism
2734 8411 ' 23A CCA-ACA Pro-Thr polymorphism
2735 8415 ' 23A CAG-CTG
' Gln-Leu polymorphism
2745 8446 23A TCT-TCG Ser-Ser
polymorphism
2760 8490 . 23A ATG-ACG Met-Thr
2761 8493 23A CGC-CCC Arg-Pro
_ 2763 8498 23A __ CTC-GTC Leu-Val _
2764 8502 23A ATG-ACG Met-Thr
2765 8504 23A CGC-TGC Arg-Cys
polymorphism
in frame
2766 8507 23A 12bp ins/dup
mutation
_ _____________________________________________________________________
2782 8556 23A GTG-ATG Val-Met polymorphism
2791 8583 23A + 23B CGG-CAG Arg-Gln ,
2813 8650 23A + 23B AGC-AGT Ser-Ser polymorphism
2814 8651 23A + 23B GGG-AGG Gly-Arg polymorphism
26
CA 02872385 2014-11-27
2815 8657 23A 4- 23B c del=gcc-gAcc Ala-Ala
frameshift
2826 8688 23B 4- 23C ATC-ACC Ile-Thr
_________________________________________________________________________ _
2888 8873 230 CGC-GGC Arg-Gly
polymorphism
2893 8890 23C TCC-TCG Ser-Ser
polymorphism
2900 8909 230 CAG-TAG Gln-X
termination
2905 8924 230 GTC-ATC Val-Ile
polymorphism
_________________________________________________________________________ _
' 2921 8973 230 CAT-COT His-Pro
2966 9109 24 GAG-GAC Glu-Asp
polymorphism
2971 9124 24 GCT-GCC Ala-Ala
polymorphism
2972 9125 24 GAC-AAC ¨ Asp-Asn *
polymorphism
4 , ________________________________________
in frame _
2978 9142 24 tie del del of Phe
deletion
2985 9164 2:5 AGA-GGA Arg-Gly
2988 9175 25 GCG-GCA Ala-Ala
polymorphism
2993 9189 25 CTG-CCG Leu-Pro
probable path.
1
3001 9213 25 TGG-TAG Trp-X
termination
3008 9233 25 GTG-CTG Val-Leu
in frame
3012 9245 25 18bp del
deletion
____________________________________________ ¨ ____________ _ _________
3016 9258 25 CAG-CGG Gln-Arg
probable path.
_________________________________________________________________________ _
27
CA 02872385 2014-11-27
' 3020 9269 25 GAG-TAG Glu-X
termination '
3030 9299 25 c del=ctg-Atgc Leu-Cys frameshift
2985 9326 25 CGC-TCG Arg-Cys
,
3052 9367 25 GGC-GGT Gly-Gly
polymorphism
3064 9401 25 TTT-CTT Phe-Leu
3065 9406 25 GTTT-CC __ 1-1 Phe-Leu
polymorphism
3065 9406 2.5 GTG-GTC Val-Val
polymorphism
3066 9407 25 TTT-CTT Phe-Leu
polymorphism
__________________________________________________________________ _
__________
3090 9481 ' 26 GTC-GTT Val-Val
polymorphism
__________________________________________________________________ _
__________
3110 9541 26 CCT-CCC Pro-Pro
polymorphism
3139 9627 27 GGC-TGC Gly-Cys
3180 9751 27 TGG-TGA Trp-X termination
3193 9789 . 28 CCT-CTT Pro-Leu
3206 9827 28 - CAG-TAG Gln-X termination
_ 3219 _________ 9867 28 t del= cti-cAU _ Leu-Leu
frameshift
3223 9880 28 ACG-ACA Thr-Thr
polymorphism
3285 10064 29 GTT-ATT Val-Ile
3311 10143 30 CAT-CGT _ His-Arg _
3341 10234 30 CTT-CTC Leu-Leu
polymorphism
,
28
,
CA 02872385 2014-11-27
3348 10255 30 CGG-CGT Arg-Arg
polymorphism
frameshift after
3350 10262 31-34 2kb del
3350
3375 10334 31 GTG-ATG Val-Met ____________ I
frameshift after
IVS31-F 25de11 9
3389
3394 10391 32 CAG-TAG Gln-X
termination
34-3'UTR 5.5kb del
3474 10631 34 CAG-TAG Gln-X
termination
3509 10737 35 ACG-ATG Thr-Met
polymorphism
3510 10739 35 CTG-GTG Leu-Val
probable path.
3511 10743 35 GCG-GTG Ala-Val
3513 10748 35 CAG-TAG Gln-X
termination
3561 10893 36 AGC-AAC Ser-Asn
probable poly.
3579 10947 36 t ins=ttc-ttAt Phe-Phe
frameshift
3589 10976 36 CTG-TTG Leu-Leu
polymorphism
IVS37-10C-A intron 37 unknown
poly
_ _________________________________________
3631 11104 37 GAG-GAC Glu-Asp
3677 11241 38 ATG-ACG Met-Thr
3692 11284 38 . t ins=gge-get Gly-Gly frameshift
I _ ________ _
29
CA 02872385 2014-11-27
3692 11285 38 c ins=tea-Actc Ser-Leu frameshift
3711 11342 38 CGG-GGG Arg-Gly
franieshift
15bp del=egg- in frame
3747 11449 39 Arg-Arg
Acgg deletion
15bp del=gcg- in frame
3749 11457 39 Arg-Gln
Acag deletion
3752 11466 39 CGG-CAG Arg-Gln
IVS39 i-1G-C Ggt-Gct splice donor
139E40-25 to
72bp del
139E40+47
3370 11521 , 40 TCG-TCA Ser-Ser
polymorphism =
3780 11549 40 10bp ins=tac-t^ac Tyr-Tyr
frameshift
3781 11554 40 GAC-GAT Asp-Asp
polymorphism
3791 11584 , 40 TCG-TCC Ser-Ser
polymorphism
3794 11592 40 TGG-TAG Trp-X termination
IVS41-11C-T intron 41 unknown
poly
3818 11665 41 TAC-TAA Tyr-X termination
3820 11669 41 CAG-TAG Gln-X termination
3837 11720 41 CAG-TAG Gln-X termination
3971 12124 43 CGC-CGT Arg-Arg
polymorphism
,
,
CA 02872385 2014-11-27
3984 12163 43 TCC-TCG Ser-Ser
polymorphism
3985 12165 43 GCA-GGA Ala-Glu
3985 12168 43 GCC-GGG Ala-Gly
probable poly.
3991 12184 43 GCC-GCG Ala-Ala
polymorphism
12187 43 9bp ins in frame
complex
IVS43+14de120
splicing
,
complex
IVS43+17de118
,
splicing
_ ____________________________________________________________________
44 CAG-CAC splice acceptor
skip exon 44
_____________________________________________________________________ ¨
4010 12239 44 CAG-TAG Gln-X termination
4011 12244 44 TGG-TGA Trp-X termination
4014 12252 44 tt del¨ttt-t"gg Phe-Trp
frameshift
_____________________________________________________________________ ¨
4017 12262 44 at del=aca-ac^t Thr-Thr
frameshift
4020 12269 44 CGA-TGA Arg-X termination
4024 12281 44 GAG-TAG Glu-X termination
4027 12290 44 g ins=ggg-ggAg Gly-Gly frameshift
4031 12303 44 GGC-GAC Gly-Asp ,
4032 12307 44 CTG-CTC Leu-Leu
polymorphism
4039 12328 44 TAC-TAA Tyr-X termination
31
CA 02872385 2014-11-27
4041 12332 44 CAG-TAG Gln-X
termination
4044 12341 44 ATC-GTC Ile-Val
probable poly.
III 44 GGT-GCT splice donor
del of 4001-
4045
1111 45 CAG-CAA splice acceptor
skip exon 45
4058 12384 45 GCC-GTC Ala-Val
probable poly.
4059 12386 45 CAG-TAG Gln-X
termination
20bp ins=ggg-
4069 1241.6 45 Gly-**
frameshi ft
gA****
20bp ins=gcc-
4075 12438 45 Ala-Ala
frameshi ft
geg
,
4086 12469 45 TGT-TGA Cys-X
termination
4091 12483 45 GCA-GCG Ala-Ala
polymorphism
4101 12511 45 g ins=-ggg-geg Gly-Gly
frameshi ft
4124 12581 45 CAG-TAG Gln-X
termination
4126 12589 45 TAC-TAG Tyr-X
termination
gtt del=gagn-
4131 12601 45 Lcu-Phe
frameshift
gegtt
4135 12614 45 AGG-GGG Arg-Gly
4136 12617 45 CTG-TTG Leu-Leu
polymorphism
,
,
,
32
CA 02872385 2014-11-27
4136 12617 45 c del=ctg-Atgc Leu-Cys
frameshift
_____________________________________________ ¨ _____________ _ __________
4139 12628 45 TGG-TGA Trp-X
termination
_____________________________________________ ¨ _____________ _ __________
4145 12644 45 GTC-ATC Val-Ile
probable poly.
_____________________________________________________________ _ __________
IVS45+17insG intron 45
unknown poly
4153 12668 46 CGC-TGC Arg-Cys
,
duplication of
4168 12714 46
frameshift
23bp
_____________________________________________________________ _ __________ _
4176 12739 46 a del----cca-ccAc Pro-Pro
frameshift
_____________________________________________________________ _ _________
4189 12777 46 TCC- FTC . Ser-Phe
polymorphism
4198 12801 ' 46 del 28
frameshift
4209 12838 46 CCT-CCC Pro-Pro
polymorphism
4224 12882 46 CAG-CCG Gin-Pro
probable path.
_____________________________________________________________ _ __________
4227 12890 46 CGA-TGA Arg-X
termination
4236 12919 46 TAC-TAa/g Tyr-X
termination
4254 12973 46 CCC-CCT Pro-Pro
polymorphism
4275 13034 46 CGG-TGG Arg-Trp
probable path.
*Updated March 2001. ** is an unidentified base or amino acid.
'
III. Identification of Unique Sites Within PKD Genes
' Due to the fact that 70% of the PKD-1 gene is replicated as non-
functional homologues
with more than 95% sequence identity to PIGD-1, the identification of PKD-1
unique sites are
critical for the development of a genetic testing method. With the successful
decoding of human
33
CA 02872385 2014-11-27
genome sequences, the unique sites within the PKD genes may be identified by
comparing
genomic DNA sequences comprising a PKD gene with genomic DNA sequences
comprising a
PKD homologue. Useful databases and computer programs are known in the art
(e.g., databases
available through NCBI at www.ncbi.alm.nih.gov; and computer programs
available at
http://wwvv.ncbi.nlm.nih.gov/BLAST and DNAStar, www.dnastar.com). A unique
site refers to
a stretch of sequence within a PKD gene which shares less than or equal to 80%
(e.g., less than
or equal to 70%, or 60%, or 50% or 40% or 30% or 20% or 10%) sequence identity
to a PKD
homologue or other sequences.
Several unique sites (e.g., single copy site) have been described in Rossetti
et al., 2000,
Am. J. Hum. Genet. 68:46-63, the entirety of which hereby incorporated by
reference. A novel
unique site (5' AGO TCC AGO GCG ACT CGC TGG 3', or 5' CAG GGC CAC ACG CGC
TOG GCG 3', or their complement thereof) is identified for PKD-1 by Applicants
of the present
application. Other unique sites may be found in, for example, in U.S. Patent
Nos. 6,228,591 and
6,031,088.
The identified unique sites can be used for designing PKD-specific primers for
the
amplification of authentic PKD genes. The length of a unique site may vary
from several
nucleotides to thousands of nucleotides. Most of unique site identified
comprises less than or =
equal to 100 nucleotides, e.g., less than or equal to 50 nucleotides, or less
than or equal to 30
nucleotides. Amplification using PKD-specific primers would increase the
specificity of the
amplification reaction and reduce the amount by-products amplified from PKD
homologues.
The specifically amplified product of authentic PKD genes may be subsequently
used for
sequencing to identify allele variant, e.g., a mutant PKD gene, in an
individual or for cloning
= and/or expression for other analysis.
IV. PKD-Specific Primers Useful for the Invention
Samples to be analyzed for the presence or absence of mutations often contain
amounts
of material too small to detect_ The first step in mutation detection assays
is, therefore, sample
amplification. A preferred amplification reaction of the invention is PCR. PCR
amplification
comprises steps such as primer design, choice of DNA poIymerase enzyme, the
number of
amplification cycles and concentration of reagents., Each of these steps, as
well as other steps
34
CA 02872385 2014-11-27
involved in the PCR process affects the purity of the amplified product.
Although the PCR
process and the factors which affect fidelity of replication and product
purity are well known in
the PCR art, these factors have not been addressed, heretofore, in relation to
mutation detection
of PKD genes using the separating method of the invention, e.g., DHPLC.
Any primer which anneals, under specific stringent conditions, to a sequence
within an
authentic PKD gene, but not to a PKD homologue or other sequences is a useful
PKD-specific
primer according to the invention. Sequences of the identified unique sites
serve as the basis for
designing PKD-specific primers useful according to the invention. The primers,
according to the
subject invention, may be incorporated into a convenient kit for identifying a
PKD patient.
A. Criteria for Selecting Primers
A PKD species-specific primers preferably comprise a sequence complementary to
a
sequence located within a unique site of a PKD gene. The PKD-specific primer
may be
complementary to a unique site of a normal or a mutant PKD gene, so long as
the primer
preferably anneals to an authentic PKD gene other than a PKD homologue.
PKD species-specific primers may be selected manually by analyzing sequences
of the
unique sites identified for a PKD gene. When the sequence of a DNA fragment to
be amplified
by PCR is known, commercially available software can be used to design primers
which will
produce either the whole fragment, or any sequence within the fragment. The
melting map of a
fragment can be constructed using software such as MacMeltRTm (BioRad
Laboratories,
Hercules, Calif.), MELT (Lerman et al. Meth. Enzymol. 155:482 (1987)), or
WirilvIeltIm
(BioRad Laboratories).
It is known in the art that primers that are about 18-25 bases long and with
50% G-C
content will work well at annealing temperature at about 52-58 C. These
properties are
preferred when designing primers for the subject invention. Longer primers, or
primers with
higher G-C contents, have annealing optimums at higher temperatures;
similarly, shorter primers,
or primers with lower G-C contents, have optimal annealing properties at lower
temperatures. A
convenient, simplified formula for obtaining a rough eStimate of the melting
temperature of a
primer 17-25 bases long is as follows:
=
CA 02872385 2014-11-27
Melting temperature (Tm in C) = 4 x (# ofG + # of C) 2 x (# of A # of T)
The overall design process design consists of both long range (i.e., for the
first round
PCR) and short range primer (i.e., for the nested PCR) design. In long range
primer design, the
objective is to design primers that produce good quality PCR products. "Good
quality" PCR
products are defined herein to mean PCR products produced in high yield and
having low
amounts of impurities such as primer dimers and PCR induced mutations. Good
quality PCR
can also be affected by other reaction parameters, such as the enzyme used,
the number of PCR
cycles, the concentration and type of buffer used, temperature thermal cycling
procedures and
the quality of the genomic template. Methods for producing good quality PCR
products are
.. discussed by Eckert et al. (PCR: A Practical Approach, McPherson, Quirke,
and Taylor eds., IRL
Press, Oxford, Vol. 1, pp. 225-244, 1991).
Short range primer design should fulfill two requirements. First, it should
fulfill all the
= requirements of long range primer design and give good quality PCR,
products. In addition, it
must produce fragments that allow the DHPLC method to detect a mutation or
polymorphism
regardless of the location of the mutation or polymorphism within the
amplified fragment. For
example, large DNA fragments, having up to several thousand base pairs, can be
amplified by
PCR. If the only goal of the amplification is to replicate the desired
fragment, then there is a
large latitude in the design of primers which can be used for this purpose.
However, if the
purpose of a PCR amplification is to produce a DNA fragment for mutation
detection analysis by
DHPLC, then primers must be designed such that the fragment produced in the
PCR process is
capable of being detected, and will produce a signal, when analyzed by DHPLC.
In a preferred
embodiment of the invention, the length of an amplified product is 150-600
bps. In a more
preferred embodiment, the fragment length for DHPLC mutation detection
analysis is l50-400
bp.
There are two goals of designing short range primers. One goal for primer
design is if the
analysis is used as a "mutation analysis" test. Another goal is in analysis
for research or
diagnostic purposes, e.g., for identifying a PKD patient. "Mutation analysis"
is defined herein as
the study or analysis of DNA fragments to determine if the fragments contain
variations (i.e.,
36
CA 02872385 2014-11-27
mutations or polymorphisms) in a population and correlate that variation to
disease. It is to be
understood that, within the context of this invention, the term "mutation"
does not include a
polymorphism (e.g., normal) which is silent for the disease. When DHPLC is
used as a mutation
analysis technique, then an important aspect of the present invention is a
method for designing
primers to produce a fragment in which a putative mutation can be detected,
regardless of where
the mutation site is located within the fragment. If the mutation is known, on
the other hand,
then the primer design can be further refined so that the analysis is
optimized, i.e., the resolution
of the homoduplex and the heteroduplex peaks in DHPLC is maximized. By
improving the
resolution for the analysis of known mutations, accuracy of analysis can be
performed.
Improved resolution is required for diagnostic mutation applications.
Furthermore, with
' improved resolution, automatic identification of the positive presence of
mutation can be more
easily implemented with appropriate software and an algorithm that overlays
and comparatively
measures the peak S of the normal and mutant DNA samples.
Another method of primer design for mutation analysis applications is to
design the
primers so that the region of interest is at a lower melting domain within the
fragment. In this
case the primers are preferred to be designed so that the fragment being
measured will overlap
the regions of interest as the analysis is performed traveling down the exon.
In these cases, the
temperature difference between the higher melting domain and the lower melting
domain is
preferred to be greater than 5 C and most preferred to be greater than 10 C.
Once the mutation of interest is identified, primers can be redesigned for
diagnostic or
clinical applications. In these cases, the mutation is preferably located
within 25% or 25 bases of
the end whichever is closer to the end. The other end of the fragment contains
a higher melting
domain of preferably 5 C, more preferably 10 C higher, and most preferably 15
C higher than
the lower domain where the mutation is located. If the primer selection does
not result in a high
melting domain on the opposite end of the fragment, then a G-C clamp can be
applied to increase
the melting temperature at the desired end (e.g., an A-T rich end) (Myers et
al., 1985, Nucleic
Acids Res. 13:3111). G-C clamping is a technique in which additional G or C
bases are included
on the 5' end of one or both of the primers. The polymerase enzyme will extend
over these
additional bases incorporating them into the amplified fragment thereby
raising the melting
temperature of the end(s) of the fragment relative to that in the vicinity of
the mutation. For
37
CA 02872385 2014-11-27
example, in cases where the mutation is in the center of the amplified
fragment and the length is
less than 100 bp and the melting profile is flat, or in cases where the
mutation in a high melting
region of the fragment and a higher melting region is in effect a G-C rich
region, a G-C clamp
may be necessary. In these cases, proper primer selection will result in a
fragment in which the
mutation can be detected. The size of the G-C clamp can be up to 40 bp and as
little as 4 or 5 bp.
The most preferred G-C clamp for mutation detection by DHPLC is 10 to 20 bp.
If it is not possible to design primers which will produce, upon PCR
amplification,
domains having a constant melting range or domains within a fragment which are
sufficiently
close in Tm, then it may be necessary to lower the Tm of a domain of interest
for successful
mutation detection by DHPLC. This can be done, for example, by substituting
dGTP with the
analog 7-deaza-2'-dGTP which is known to effectively lower the melting
temperature of G-C
base pairs (Dierick et al.,1993, Nucl: Acids Res. 21:4427). If it is necessary
to raise the Tm of
the domain, then 2, 6-aminopurine can be used in place of dGTP in the PCR
amplification.
In a most preferred embodiment, the primers are selected so that the mutation
is located
in a "lower melting" domain of the fragment. However, a mutation can also be
detected by
DHPLC in a high melting domain of the fragment either if the high melting
domain does not
have a melting temperature that is too different from other domains in the
fragment or if a higher
column temperature is used that is optimized for the higher melting domain of
the fragment.
The long range primer design described above can be further refined by local
primer
design in which several other factors should be considered. For example,
primers with non-
template tails, such as universal sequencing primers or T7 promoters, may need
to be avoided.
The preferred primer has a Tm of about 56 C. The difference in Tm between the
forward and
reverse primers is preferably about 1 C. The difference in Tm between primer
and template is
preferably 25 C. The 3'-pentomer of each primer is preferably be more stable
than AG -=---6
kcal/mol (i.e., more negative). Any possible primer dimers are preferably be
less stable than the
3'-pentomer by at least 5 kcal/mol (i.e., 5 kcal more positive). Any primer
self annealing loops
are preferably to have a Tm of less than 12 C. Primers are preferably be of
high purity without
failure sequences. To avoid degradation, storage in Tris-HCl (pH 8.0) buffer
is preferable to
pure water.
38
CA 02872385 2014-11-27
In some embodiments, it is more convenient to directly separate a tong
fragment, e.g., an
exon, of up to 5 kb (e.g., up to 4 kb, or up to 3kb, or up to 2 kb, or up to
1kb) for mutations.
Such long fragments generally contain multiple melting temperature domains.
Double-stranded
DNA fragments melt in a series of discontinuous steps as different regions
with differing thermal
stabilities which denature in response to increasing temperature. These
different regions of
thermal stability are referred to as "domains", and each domain is
approximately 50-300 bp in
length. Each domain has its own respective Tm and will exhibit thermodynamic
behavior which
is related to its respective Tm. The presence of a base mismatch within a
domain will destabilize
it, resulting in a decrease in the Tm of that domain in the heteroduplex
relative to its fully
hydrogen-bonded counterpart found in the homoduplex. Generally the presence of
a base
' mismatch will lower the Tm by approximately 1-2 C.
In accordance with the preferred embodiments, optimal results have been
obtained using
primers which are 18-51 in length and DNA sequence to the primers with SEQ ID
NOs. 3-49
(Table 3 and Table 4). However, one skilled in the art will recognize that the
length of the
primers used may vary. For example, it is envisioned that shorter primers
containing at least 15,
and preferably at least 17, consecutive bases of the nucleotide sequences of
these primers SEQ
ID NOs. 3-49 may be suitable. The exact upper limit of the length of the
primers is not critical.
However, typically the primers will be less than or equal to approximately 60
bases, preferably
less than or equal to 50 bases. Further still, the bases included in the
primers may be modified as
is conventional in the art, including but not limited to, incorporating
detectable labels such as
biotin, or fluorescent labels.
=
Table 3 Examples of useful PKD-1 specific primers*
SEQ ID NO. Primer Name Primer Sequence
1X1F 5' CGT CGC TCA GCA GCA GGT CG 3'
1X1R 5' CGT CCT GCT TCC CGT CCC G 3' = '
1X2F 5' GCG GCC CGC CGC CCC CGC CGT TGG GGA
TGC TGG CAA TGT GTG 3'
39
CA 02872385 2014-11-27
1X2R 5' GGG AU COG CAA AGC TGA TG 3'
1X3F 5' TTC CAT CAG CTT TGC CGA AT 3'
1X3R 5' ATC TOG TCT CAA GCC TOG AAG
1X4F 5' GCC CCG CGC CCG TCC CGC CGC CCC CGC CGA
GAC CCT TCC CAC CAG ACC T 3'
1X4R 5' CGC CCC CGC CCG TGA GCC CTG CCC AGT GTC
T 3'
1 X5AF 5' GCG GCC CGC CGC CCC CGC CGG AGC CAG
GAG GAG CAG AAC CC 3'
1X5AR 5' CAG AGO GAC AGO CAG GCA AAG 03'
1X53F 5' GCC CCC GCC GCC CAG CCC TCC AGT GCC 13'
1X5BR 5' ATC GCT ATG TGC TGC CTG GG 3'
1X5CF 5' CCG AGO TOG ATG CCG CTG 3'
1X5CR 5' GAA GGG GAG TGG GCA GCA GAC 3'
1X6F 5' CAC TGA CCG TTG ACA CCC TCG 3'
1X6R 5' TGC CCC AGT GCT TCA GAG ATC 3'
1X7F 5' GOA GTG CCC TGA GCC CCC 13'
1X7R 5' CCC CTA ACC ACA GCC AGC G 3'
1X8F 5' TCT GTT COT CCT GGT GTC CTG 3'
1X8R 5' GCA GGA GGG CAG GTT GTA GAA 3'
CA 02872385 2014-11-27
1X9F 5' GCG GCC CGC CGC CCC CGC CGG CIA GGG
GGA GTC TGG GCT T 3'
1X9R 5' GAG GCC ACC CCG AGT CC 3'
1X1OF 5' GTT GGG CAT CTC TGA COG TO 3'
1X1OR 5' CGC CGC CCC CGC CCG GGA AGG TGG CCT
GAG GAG AT 3'
1X11AF 5' GCG GCC CGC CGC CCC CGC COG GGG TCC
ACG GGC CAT G 3'
1X11AR 5' AAG CCC AGC AGC ACG GTG AG 3'
1X11BF 5' CCG CCG CCC CCG CCG CTG CCC TGC CTG TGC
CCT G 3'
1X11BR 5' GCC CCG CGC CCG TCC CGC CGC CCC CGC CCG
TTC CAC CAC CAC GTC CAC CAC 3'
1X11CF 5' GTG GTG GAC GTG GTG GTG GAA 3'
1X11CR 5' GGC TGC TGC CCT CAC TGG GAA 3'
,1X12F 5'TAA GGG CAG AGT CCT CCA CAG 3'
1X12R 5'CCA CCC CCG CCC ACC TAC TGA G 3'
1X13F 5' GCG GCC CGC CGC CCC CGC CGT GGA GGG
AGG GAC GCC AAT C 3'
1X13R 5' GAG GCT GGG OCT GGG ACA A 3'
1X14F 5' CCC GOT TCA CTC ACT GCG 3'
41
CA 02872385 2014-11-27
1X14R 5' CCC CCG CCC GCC GTG CTC AGA GCC TGA
AAG 3'
1X15AF 5' GGC GGG GGG CTT CTG CCG AGC GGG TGG
GGA GCA GGT GG 3'
1X1SAR 5' CGC CGC CCC CGC CCG GCT CTG GGT CAG
GAC AGO GGA 3'
1X15BF 5' CGC CTG GGG GTG TTC TTT 3'
1 X1 5BR 5' ACG TGA TGT TGT CGC CCG 3'
1X15CF 5' GCC CCC GCC GGG GCG CCC CCG TGG TOG
TCA GC 3'
1X15CR 5' CAG GCT GCG TGG GGA TGC 3'
1X151iF 5' CTG GAG GTG CTG CGC G1 -I 3'
IXI5DR 5' CGC CCC CGC CCG CTG GCT CCA CGC AGA
TGC 3'
1X15EF 5' COT GAA CAG GGC GCA TTA 3'
1X15ER 5' CCC CCG CCC GCiC AGC AGA GAT urr GTT
GGA C 3'
1X15FF 5' CCG CCG CCC CCG CCG CCA GGC TCC TAT CTT
GTG ACA 3'
1X15FR 5' TGA AGT CAC CTG TGC TGT TGT 3'
1X15GF 5' CTA CCT GTG GGA TCT GGG G 3'
1X15GR 5' TGC TGA AGC TCA CGC TCC 3'
42
CA 02872385 2014-11-27
1X15HF r 5' GGG CTC GTC GTC AAT GCA AG 3'
1X15HR 5' CGC CGC CCC CGC CCG CCG CCC ACC ACC TGC
AGC CCC TCT A 3'
1X15IF 5' GCG GCC CGC CGC CCC CGC CGC CGC CCA
GGA CAG CAT CTT C 3'
1X151:12. 5' CGC TGC CCA GCA TGT TGG 3'
1X15JF 5' GGC CGG CAG CGG CAA AGG CTT CTC 3'
1X15JR 5' GCC CAG CAC CAG CTC ACA T 3'
1X15KF 5' CGA GCC ATT TAC CAC CCA TAG 3'
1X15KR 5' GGC AGC CAG CAG GAT CTG AA 3'
1X15LF 5' CTG TGG GCC AGC AGC AAG GTG 3'
1X15LR 5' CCT GAA CCT CCA GCA CCA GCG 3'
1X151v1F 5' AUG TCC AGO GCG ACT CGC TGG 3'
1X15114R 5' CAG GGC CAC ACG CGC TGG GCG 3'
1X15NF 5' TTG GAG GCC CAC G'TT GAC CTG 3'
1X15NR 5' CCC CCG CCC GCA TOG GTG TGG ACG GOT
GAG G 3'
1X16F 5' TAA AAC TGG ATG GGG CTC IC 3'
1X16R 5' GGC CTC CAC CAG CAC TAA 3' =
1X17F 5' GGG TCC CCC AGT CCT TCC AG 3'
43
CA 02872385 2014-11-27
1X17R 5' TCC CCA GCC CGC CCA CA 3'
1X18F 5' GCC CCC TCA CCA CCC CTT CT 3'
1X18R 5' TCC CGC TGC TCC CCC CAC GCA 3'
1X19F 5' GAT GCC GTG GGG ACC GTC 3'
1X19R 5' GTG AGC AGG TGG CAG TCT CG 3'
1X2OF 5' CCA CCC CCT CTG CTC GTA GGT 3'
1X2OR 5' GOT CCC AAG CAC GCA TGC A 3'
1X21F 5' TGC CGG CCT CCT GCG CTG CTG A 3'
1X21R 5' GCG GGC AGG GTG AGC AGG TGG GGC CAT CC
3'
1X22F 5' GAG GCT GTG GGG GTC CAG TCA AGT GO 3'
1 X22R 5' AGG GAG GCA GAG GAA AGG GCC GAA C 3'
1X23AF 5' COT CCC GCC TGC ACT GAC CTC ACG CAT GT 3'
1X23AR 5' CGG CCC GCC GCC CCC GCC CGG CCA AAG
GGA AAG GGA TTG GA 3'
1X23BF 5' CCG CGG AGC CTG CTG TGC TAT 3'
1X23BR 5' CCG CCG CCC CCG CCC OCT TGG TGG AGA
CGG TGT AGT TGC 3'
1 X23CF 5' TCC AAT CCC ITT CCC TTT GGC 3'
X23CR 5' CAG CAG CCC ATG AAA CAG AAA G 3'
44
CA 02872385 2014-11-27
1X24F 5' TAT GCT TTC AGG CCC GTG GCA 3'
1X24R 5' AGA GCC CAT ACC CGG TCC AGT CC 3'
1X25F 5' GGA CTG GAC CGG GTA TGG GCT CT 3'
1X25R 5' CCC CCG CCC GCA CCC AGG CCC TCC TCG ACT
C 3'
1X26F 5' CCC CCG CCG CTG GGT GGG CTC GGC TCT ATC
3'
1X26R 5' TGG TAG CGA TGC TCA CGT CAC TT 3'
1X27F 5' CAG GCC AAA GCT GAG ATG ACT TG 3'
1X27R 5' AGA GGC GCA GGA GGG AGG TC 3'
1X28F 5' CCC TCT GCC CCC GCA TTG 3'
1X28R 5' AAG CGC AAA AGG OCT GCG TCG 3'
1X29F 5' GGC CCT CCC TGC CII CTA GGC 03
1X29R 5' CCG TGC TOT GTG GAG GAG AG 3'
1X30F 5' OCT OTT OCT GCC CAG CCC TTC 3'
1X3OR 5' CU CCC GAG CAG CCT TTG GTG 3'
1X31F 5' CTG AGO TGC COO CCG CTG AC 3'
1X31R 5' AGG ACC CCC AGC CCA GCC CA 3'
1X32F 5' CIT GGC GCA GCT TGG ACT 3'
1X32R 5' ACA CCC AGC AAG GAC ACG CA 3'
CA 02872385 2014-11-27
1X33F 5' TGT GAC ACA TCC CCT GOT AC 3'
1X33R " 5' GCA AGO GIG AGC TTC AGA GC 3'
1X34F . 5' GCC CCG CGC CCG TCC CGC CGC CCC CGC CCG
ACC CTA TGC CTC CTG TAC CTC 3'
1X34R 5' CCC CTC CTC TOG CAA TCC 3'
3 1X35F 5' TGG CTG CAA CTG CCT CCT GO 3'
4 1X35R 5' AAG CAG AGA CAG ACC TOT GAG AG 3'
1X36F 5' GCC CCC GCC OCT CTC ACA GGT CTG TCT CTG
CTT C 3'
6 1X36R 5' GGC CTG TAG CCT ACC CCT GO 3'
7 1X37F 5' GGA CCC CTC TGA AGC CAC C 3'
-8 1X37R 5' GGG AGG TGG GAG ACA AGA GAC 3'
9 1X38F 5' AAA GCC CTG CTG TCA CTG TGG 3'
1X38R 5' AAC TAA AGC CCA GAA GAC AGA CC 3'
11 1X39F 5' AAC TOT CTG CCC CAG AAC ATC 3'
12 1X39R 5' CTA AAG GCT GCT CTC TCA ACA AG 3'
13 1X4OF 5' ACT CCT OTT GGG TTT TGA TGA G 3'
14 1X4OR 5' GAG AAC TAC TCC CTT GTC CTT GG 3'
1X41F ---- 5' ACG CCA AGG ACA AGO GAG TAG TTC 3'
46
CA 02872385 2014-11-27
16 1X41R 5' TGG OCT CCT GGC TOG TGA CTG C 3'
17 1X42F 5' GCG GCC CGC CGC CCC CGC CGC TAC TGA CCC
GCA CCC TCT G 3'
18 1X42R 5' GCT GCG AGO GGT GAG ACG 3'
19 1X43F 5' GCG GCC CGC CGC CCC CGC CGC GTC CCT CCC
GCC CTC CTG ACC 3'
20 1X43R 5' GCC CCC GCC OCT GCG GAC GAG AAA TCT
GTC TGC TTG 3'
21 1X44F 5' CAG GGC TGC AAG CAG ACA GA 3'
22 1X44R 5' CTG AGC TAA GAC GCC CTC CC 3'
23 1X45F 5' CTG TAC GCC CTC AG! GUT GTC 3'
24 1 X45R 5' GGC ACA GGG GCT CAG TCA GTC 3'
25 1X46AF 5' GGA CTG ACT GAG CCC CTG TGC 3'
26 1X46AR 5' ACT CGG TCA AAC TOG GTG AG 3'
27 1X46BF 5' CAA GOT GTG AGC CTG AGC CC 3'
28 I X46BR 5' COG TGT CCA CTC CGA CTC CAC 3'
*All primer sequences are denoted in the 5'-3' direction. The first number in
the name
denotes the PKD gene number (IX I5AF). The Letter 'X' signifies the word exon
(1X15AF).
The third number after the 'X' denotes the exon number (1X15AF). The character
after the exon
number represents the identity of the exon fragment (1X15AF). The last letter
indicates the
direction of the primer as either forward or reverse (1X15AF).
47
CA 02872385 2014-11-27
Table 4 Examples of useful PICD-2 specific primers*
SEQ ID NO. Primer Name - Primer Sequence
29 2X1AF 5' CCG CCC CCG CCG CGC GCC GGA CGC CAG
TGA CC 3'
2X1AR 5' CCT GCC GGG AGC ACG ACG AG 3'
30 2X1BF 5' GCC CCC GCC GCC GCG GCC TCC CCT TCT
CCT 3'
2XIBR 5' CTG GGC TGG GGC ACG GCG GO 3'
2X1CF 5' GGG GGC TAC CAC GGC GCG GGC 3'
31 ¨2X1CR 5' CGG CCC GCC GCC CCC GCC CGC GGC CGT T
GGT TCG TGC ATC TG 3'
32 2X2F 5' GCC CCC GCC GAA ATG ATA TCT UT (
TTC TTC A 3'
33 2X2R 5' CCC CCG CCC GAA CTT TCC CAT TAG TGC A
3'
2X3F 5' TTG GGG COT TCA TTT GGA TC 3'
34 2X3R 5' CGC CGC CCC CGC CCG TOT GAT AGA GAG (
CU TCA 3'
35 2X4F 5' CCG CCG CCC CCG CCG CTT TTT CAA AGA T
TTC CTT TGC 3'
36 2X4R 5' TAT CAC CGA GTG CCA ATG AG 3'
37 2X5F 5' CCG CCG CCC CCG CCG GCC TCA AGT GTT C
CTG AT 3'
2X5R 5' ACC ACA CAG AAA TAG GAG GO 3'
2X6F 5' TTG TTA TTG TTT TAA TTG TTC TTA 3'
48
CA 02872385 2014-11-27
38 2X6R 5' CCC CCG CCC GTT GTA GAA TAG AAT AGO
TTT GG 3'
39 2X7F 5' GCC CCC GCC GTT GOT GAA GAA AAA TAT
AGT CA 3'
40 2X7R 5' CGC CGC CCC CGC CCG TGG AAC TCA TTT T
TTA AAG A 3'
41 2X8F 5' GCG GGG GCG GCG GGC COT TTT ATT ATA C
AGT CAC ACC 3'
2X8R 5' CIA CTC TGA CTA AAT TTT TCT TCT T 3'
2X9F 5' TIT GGT FIT GTA ITO TGG TO 3'
,2X9R 5' AAG GAT TTA CGA AGT TTA AAT TG 3' .
42 2X1OF 5' GCC CCC GCC OCT TCC UT AAT TTT TGC CC
' 3'
43 2X1OR 5' CGC CGC CCC CGC CCG GAA ACA ATG CTC A
TTA TGT CAG 3'
44 2X11F 5' CCG CCG CCC CCG CCG AAA CCA AGT CUT
TTT TTT CTC 3'
2X11R 5' AGA ACC TCA QUA AGC ATG ATT 3'
45 212F 5' CCG CCG CCC CCG CCG GAT GAA TGT TAT C-
TAT CCT CTC 3'
2XI2R 5' TAG GTA CCA. AAT CAA ATC CO 3'
2X13F 5' GTC TCA GTG TTC TGC TCC TC 3'
46 2X13R 5' CGC CGC CCC CGC CCG GCA AAT TCT GCC A
TCC Ill A 3'
47 2X14F 5' GCC CCC. GCC GTT TGT CCC TCT GTA CTG TG
3'
2X14R 5' AAA TAC AAC TOT CAG CAA CAT A 3'
49
CA 02872385 2014-11-27
48 2X15F 5' CCG CCC CCG CCG TGA CCC CCA ACA CCA G
TC 3'
49 2X15R 5' COG CCC GCC GCC CCC GCC COG GAC AGC C
TTC CTC ACT T 3'
*All primer sequences are denoted in the 5'-3' direction. The first number in
the name
denotes the PKD gene number QX15R). The Letter 'X' signifies the word exon
(2X15R). The
third number after the 'X' denotes the exon number (2X15R). The last letter
indicates the
direction of the primer as either forward or reverse (2X1511).
B. Primer Combinations Useful for PKD-specific Amplification
The specifically amplified product can be generated by using one or more PKD-
specific
primers. Preferably, both primers used to generate one amplified product are
PKD-specific
primers. However, one PKD-specific primer can be used in combination with
another non PKD-
specific primer which is not complementary to a unique site of a PKD gene. The
non PKD-
.. specific primer is preferably designed according to the same criteria
described above herein for
the PKD-specific primers and is preferably to be completely complementary to a
sequence other
then a unique sequence in a PKD gene. A non PKD-specific primer may also be
used as a
control primer included in the amplification reaction to generate a control
product.
Optimal results may be obtained by using one forward and one reverse primer
listed in
Table 4 and Table 5, although other combinations may also be used. In a
preferred embodiment,
a primer pair is selected so that the length of an amplified product is 150-
600 bps. In the most
preferred embodiment, a primer pair is selected so that the amplified fragment
length for DF,IPLC
mutation detection analysis is 150-400 bp.
C. Primer Synthesis
Methods for synthesizing primers are available in the art. The oligonucleotide
primers of
this invention may be prepared using any conventional DNA synthesis method,
such as,
phosphotriester methods such as described by Narang et al. (1979, Meth.
Enzymol., 68:90) or
Itakura (U.S. Pat. No. 4,356,270), or and phosphodiester methods such as
described by Brown et
al. (1979, Meth. Enzymol., 68:109), or automated embodiments thereof, as
described by Mullis
CA 02872385 2014-11-27
et al. (U.S. Pat. No. 4,683,202). Also see particularly Sambrook et al.(1989),
Molecular
Cloning: A Laboratory Manual (2d ed.; Cold Spring Harbor Laboratory:
Plainview, N.Y.) .
V. Preparing Template for Amplification Reaction
Any sample comprising a nucleic acid comprising the entire or a portion of SEQ
ID NO.
1 or 2 or their variants (e.g., polymorphism forms or mutant forms) may be
used to as template
for amplification reaction of the present invention. Useful templates,
according to the invention,
=
include, but are not limited to, genomic DNA preparation, total RNA
preparation, crude cell
lysate and tissue sample.
It's preferred to use genomic DNA as template for PKD-specific amplification
of the
subject invention. While it is envisioned that crude cell lysate or tissue
sample may be used, one
skilled in the art will recognize that any non-DNA material present in the
sample may interfere
with the polyrnerase reaction or subsequent analysis.
=
Genomic DNA can be isolated from tissue samples or cells. Preferably, the
genomic
DNA used as template for the invention is isolated under conditions which
preclude degradation
and contamination. Tissue samples or cells may be digested with a protease so
that there is
likely to be little or no DNAase. activity. The digest is extracted with a DNA
solvent. The
extracted genomic DNA may be purified by, for example, dialysis or
chromatography. Suitable
genomic DNA isolation techniques are known in the art, for example, as
described in Current
protocols in molecular biology, Ausubel et al., John Weley & Sons, Inc., 1997.
Preferably, genomic DNA or cDNA is extracted from cell lysate of tissue
samples taken
from an individual and used as template for PKD amplification. Collecting a
tissue sample also
includes in vitro harvest of cultured human cells derived from an individual's
tissue or any
means of in vivo sampling directly from a subject, for example, by blood draw,
spinal tap, tissue
smear or tissue biopsy. Optionally, tissue samples are stored before analysis
by well known
storage means that will preserve a sample's nucleic acids in an analyzable
condition, such as
quick freezing, or a controlled freezing regime, in the presence of a
cryoprotectant, for example,
dirnethyl sulfoxide (DMSO), glycerol, or propanediol-sucrose. Tissue samples
can also be
51
CA 02872385 2014-11-27
pooled before or after storage for purposes of amplifying them for analysis.
In some
embodiments, the sample contains DNA, tissue or cells from two or more
different individuals.
Any human tissue containing nucleic acids can be sampled and collected for the
purpose
of practicing the methods of the present invention. A most preferred and
convenient tissue for
collecting is blood. No patient preparation is necessary prior to blood draw.
No medications are
known to interfere with sample collection or testing. Usual aseptic techniques
and avoidance of
contamination are necessary.
Preferably, DNAs are extracted from blood on the day it was drawn. Blood is
preferred
to stored at room temperature (72 F or 25 C) before use. However, whole blood
may be stored
for short periods at 4 C but room temperature is recommended. Whole blood
specimens may be
stable for 48 hrs. After this time hemolysis may compromise DNA recovery and
integrity. The
optimal amount of blood for DNA extraction for the PCR assay is preferred to
be more than 5
ml, e.g., more than 10.0 ml.
VI. PCR Amplification Using PKD-specific Primers
The subject invention provides a method of mutation analysis of a target
nucleic acid
comprising SEQ ID NO. 1 or 2 or their variants by amplifying the DNA from a
sample
comprising the target nucleic acid in a polymerase chain reaction and
detecting in a specifically
amplified product the presence or absence of a mutation in the target nucleic
acid.
Amplification may be carried out by means well known in the art, for example,
polymerase chain reaction (PCR), transcription based amplification (reverse
transcription),
strand displacement amplification (see Current Protocol in Molecular Biology).
Preferably, the
amplification is carried out by PCR, such as described by Mullis (U.S. Patent.
No. 4,683,202).
PCR makes possible the amplification (replication) of minute samples of DNA or
other
nucleic acids of any base pair length (size) by taking advantage of highly
selective enzymes
called DNA polymerases, to extend small DNA strands called "primers" along a
"template".
The minute DNA sample serves as the template. PCR reproduces the complementary
sequence
of deoxynucleotide triphosphate (dNTP) bases present in the template or any
chosen portion
52
=
CA 02872385 2014-11-27
thereof. The PCR is commonly used in conjunction with diagnostic techniques
where, for
example, a DNA sample having a concentration below the limit of detection is
amplified by the
PCR process, and the larger amount so obtained is subsequently analyzed.
Apparatus for performing PCR amplifications, e.g. Air Thermo Cycler (Idaho
-- Technologies) and reagents are commercially available from numerous
sources, e.g. Perkin-
Elmer Catalog "PCR Systems, Reagents and Consumables" (Perkin-Elmer Applied
Biosystems,
Foster City, Calif.).
PCR is typically run in a buffer at pH 5-8. The buffer contains a double
stranded DNA
sample to be amplified, a forward primer, a reverse primer, magnesium (e.g.,
as MgCl2), and the
-- four deoxynucleotide triphosphates (dATP, dTTP, dCTP, and dGTP) generally
referred to as
"dNTPs", the building blocks of DNA. The reaction mixture is heated to a
temperature (e.g., >
90 C) sufficient to denature the DNA sample, thereby separating its two
complimentary nucleic
acid strands. Alternatively, the DNA may be denatured enzymatically at ambient
temperature
using a helicase enzyme. If denaturing is effected by heat and a thennostable
DNA polymerase
-- is used, the DNA polymerase is added before the reaction is started. Other
denaturing conditions
are well known to those skilled in the art and are described in U.S. Patent
No. 5,698,400. DNA
polymerases are commercially available from a variety of sources, e.g. Perkin-
Elmer Applied
Biosystems, (Foster City, Calif) and Stratagene (La Jolla, Calif.).
The primer sequence is designed to be complimentary to an identified portion
of the
-- denatured DNA strands to be replicated by PCR. Upon cooling the reaction to
an appropriate
annealing temperature, each of the primers anneals to its complimentary base
sequence in each
strand of the denatured DNA sample to be replicated. Heated to about 70 C in
the presence of
the DNA polyrnerase, the 4 dNTPs and Mg2+, replication extends the primers
from their 3'-ends
by adding complimentary dNTPs along the length of the strand. dNTPs are
commercially
-- available from a variety of sources, e.g. Pharmacia (Piscataway, N.J.). By
repeating this process
numerous times, a geometric increase in the number of desired DNA strands is
achieved in the
initial stages of the process or as long as a sufficient excess of reagents
are present in the reaction
medium. Thus, the amount of the original DNA sample is amplified.
53
CA 02872385 2014-11-27
The amount of polymerase must be sufficient to promote DNA synthesis
throughout the
predetermined number of amplification cycles. Guidelines as to the actual
amount of polymerase
are generally provided by the supplier of the PCR reagents and are otherwise
readily
determinable by a person of ordinary skill in the art. Preferably, a DNA
polymerase with proof-
reading activity is used.
The amount of each primer must be in substantial excess of the amount of
target DNA to
be amplified. The amount of primer needed for the reaction mixture can be
estimated by one
skilled in the art in terms of the ultimate number of amplified fragments
desired at the conclusion
of the reaction.
To prevent false positive results, one skilled in the art will recognize that
the assays
should include negative controls as is conventional in the art. For instance,
suitable negative
controls may contain no primer or no DNA (i.e. "water controls"). To prevent
false negative
results, positive controls are provided by the control primers (see below).
A. Optimization of PCR Conditions
Successful specific amplification, e.g., an amplification which produces
maximal amount
of specifically amplified products and minimal amount of non-specifically
amplified products,
according to the invention, depends in great measure on the specific annealing
of the PKD-
specific primers to the corresponding matched template. If the primer anneals
non-specifically to
many different sequences in the reaction mixture, the amplification process
will not be specific.
Although it is unlikely in most of the embodiments to avoid any non-specific
annealing or non-
specific amplification, it is desirable to optimize the PCR amplification
reaction condition so to
reduce the non-specific amplification while increase the specific
amplification.
In addition, PCR induced mutations, wherein a non-complimentary base is added
to a
template, are often formed during sample amplification. Such PCR induced
mutations make
mutation detection results ambiguous, since it may not be clear if a detected
mutation was
present in the sample or was produced during the PCR process. Applicants have
recognized the
importance of optimizing PCR sample amplification in order to minimize the
formation of PCR
54
CA 02872385 2014-11-27
induced mutations and ensure an accurate and unambiguous analysis of putative
mutation
containing samples.
B. Controlling the Specificity of PK.D-specific Annealing of PKD-
specific Primers.
The degree of fidelity of replication of DNA fragments by PCR depends on many
factors
which have long been recognized in the art. Some of these factors are
interrelated in the sense
that a change in the PCR product profile caused by an increase or decrease in
the quantity or
concentration of one factor can be offset, or even reversed by a change in a
different factor. For
example, an increase in the enzyme concentration may reduce the fidelity of
replication, while a
decrease in the reaction temperature may increase the replication fidelity. An
increase in
magnesium ion concentration or dNTP concentration may result in an increased
rate of reaction
which may have the effect of reducing PCR fidelity. A detailed discussion of
the factors
contributing to PCR fidelity is presented by Eckert et al., (in PCR: A
Practical Approach, 1991,
McPherson, Quirke, and Taylor eds., IRL Press, Oxford, Vol. I, pp. 225-244);
and Andre, et. al.,
(1977, GENOME RESEARCH, Cold Spring Harbor Laboratory Press, pp. 843-852).
Thus,
availability of a product profile of the PCR process, makes possible the
optimization of PCR
conditions to improve results in a highly efficient manner.
In PCR amplification, the specificity of the annealing is most important in
the first few
cycles. The remaining cycles only serve to expend the pool of template which
is amplified in the
first few cycles. The specificity of primer annealing to template is
controlled by the ionic
strength (primarily the K+ concentration) of the buffer, the Mgz+
concentration (which is bound
= to dNTPs and therefore affected by the amount of dNTPs), and the
annealing temperature of each
cycle of the amplification. In preferred embodiments, the dNTP concentrations
are 50 nM,
preferably 100 nM, more preferably 200 riNI.
Conditions for specific annealing of primers to particular template targets
must be ,
determined empirically, usually by varying the annealing temperature in
several degree
increments and comparing the specificity and sensitivity of the amplification
process by agarose
gel electrophoresis (See Current Protocol in Molecular Biology, supra).
CA 02872385 2014-11-27
Because a unique region to which a PKD-specific primer complement to may
differ from
a homologue sequence only by a few nucleotides, sometimes by only one
nucleotide, the
specificity of the amplification reaction needs to be tested for each PKD-
specific primer used in
the reaction.
The formula for calculating primer annealing temperature provided above is
only a rough
guide, successive trials at different annealing temperatures is the usual way
to optimize this
important parameter in the PKD-specific amplification reaction. Apparatus are
available for
simultaneous testing of different annealing temperatures of particular primer-
template pairs,
which enables the optimal annealing temperature to be determined rapidly and
reliably (e.g.,
Robocycler Gradient Temperature Cycler, Cat # 400864, Stratagene; Eppendorf
mastercycler
gradient, Cat # 5331 000.045, Brinkmann Instruments, Inc. Westbury, NY).
In some embodiments, the target sequences are amplified at an annealing and
extending
temperature that is between 1 C and 10 C higher than the Tm for the primer
pair. Although
amplification at this temperature is inefficient, any primer extension that
occurs is target specific.
Consequently, during the high temperature cycle(s), the sample is enriched for
the particular
target sequence and any number of cycles, i.e., 1-15 enhances product
specificity. The annealing
temperature may be then decreased to increase amplification efficiency and
provide a detectable
amount of PCR product. Or a.nested amplification reaction may be performed
using the
amplified product from the first PCR reaction as template (see below).
Alternatively, one can simultaneously run a set of reactions at a constant
temperature but
vary the concentration of KC1 or MgCl2 or add variable amounts of a denaturant
such as
formamide (e.g., 0, 2, 4, 6%), DMSO (1-10%) to define the optimum conditions
for generating a
high yield of specific product with a minimum of nonspecific products.
In one embodiment, a pair of primers comprising at least one selected from the
group
consisting of SEQ ID NOs. 3-49 is used in the amplification reaction mixture.
The orientation of
the two primers is opposite to allow the generation of one or more
specifically amplified product.
In some embodiments of the invention, when primers used for PKD-specific
amplification are selected from SEQ ID NOs. 3-49, AmpliTaq Gold DNA polymerase
with
56
CA 02872385 2014-11-27
GeneAmp PCR buffer II and MgCl2 solution and rTth DNA polymerase XL & XL
buffer II pack
from Perkin Elmer, and TaqPlus Precision PCR system from Stratagene were used.
PFUTurbo TM is another high fidelity DNA polymerase having greater proof
reading provided by
Stratagene.
In other embodiments, an annealing temperature of above 65 C (e.g., 68-72 C)
is used for
PKD-specific amplification using primers selected from SEQ ID NOs. 3-49.
In general, it is preferred but not essential that the DNA polymerase is added
to the
amplification reaction mixture after both the primer and template are added.
Alternatively, for
example, the enzyme and primer are added last or the reaction buffer or
template plus buffer are
added last. It is generally desirable that at least one component that is
essential for
polymerization not be present until such time as the primer and template are
both present, and
the enzyme can bind to and extend the desired primer/template substrate. This
method, termed
"hot start," minimizes the formation of "primer-dimer" and improves
specificity of the
amplification.
The degree of specificity of DNA poiymerases varies with the reaction
conditions
employed as well as with the type of enzyme used. No enzyme affords completely
error free
extension of a primer. Therefore, a non-complimentary base may be introduced
from time to
time. Such enzyme related errors produce double stranded DNA products which
are not exact
copies of the original DNA sample, but contain PCR induced mutations. Other
PCR process
features, such as reaction temperature, primer annealing temperature, enzyme
concentration,
dNTP concentration, Mg2+ concentration, and combinations thereof, all have the
potential to
contribute to the degradation of the accuracy or fidelity of DNA replication
by the PCR process,
as described above herein_
C. Sensitivity of PKD-specific Amplification
The sensitivity of the PKD-specific amplification of the subject invention
depends on the
template and primers used in an amplification reaction, as well as ionic
strength and annealing
temperature of each cycle of the amplification.
57
CA 02872385 2014-11-27
When genomic DNA is used as template, as few as one or two copies of the
template
(about 3-5 pg) can be used for successful PCR amplification if the reaction
condition has been
optimized. However, it's known in the art that a higher template concentration
may increase the
specificity and efficiency of the amplification.
Shorter fragments are amplified more efficiently than longer fragments.
Preferably,
primers which generate an amplified product of less than 1 kb, more preferably
less than 600bp,
or less than 450bp in length are used to increase sensitivity of the
amplification assay.
Preferably, the sensitivity of the amplification assay is less than 100 ng
genomic DNA
template. More preferably, the sensitivity of the assay is less than 10 ng
genomic DNA template.
More preferably, the sensitivity of the assay is less than 1 ng genomic DNA
template. More
preferably, the sensitivity of the assay is less than 0.1 ng genomic DNA
template. Even more
preferably, the sensitivity of the assay is less than 0.01 ng genomic DNA
template.
D. Nested Amplification
In some embodiments of the invention, a nested amplification is performed
using
amplified products in a preceding amplification reaction as templates.
Preferably, the nested
amplification reaction is a nested PCR using PCR amplified products from a
preceding PCR
reaction as templates. In addition to optimizing the annealing temperature of
the primers,
"nested" amplification can be used to increase the specificity and sensitivity
of the PKD-specific
amplification assay.
For example, a method comprising a nested PCR involves two sequential PCR
reactions.
After multiple cycles of PCR (e.g., 10 to 40, or 10 to 30 or 10 to 20 cycles)
with the first pair of
primers comprising at least one PKD-specific primer (e.g., a PKD-specific
primer and a control
primer or two PKD-specific primers), a small amount aliquot of the first
reaction (e.g., lul of a
50 1 reaction) serves as the template for a second multiple cycles of PCR
reaction (e.g., 10 to 40,
or 10 to 30 or 10 to 20 cycles) with a new set of primers comprising at least
one PKD-specific
primer (e.g., a PKD-specific primer and a control primer or two PKD-specific
primers) that
anneal to sequences internal to, or nested between, the first pair.
58
CA 02872385 2014-11-27
Methods for designing nested primers and for performing nested PCR are known
in the
art (see Current Protocol in Molecular Biology, supra). The general criteria
for selecting primers
as described above also applies to the design of nested primers. Both nested
primers need to
anneal to sequences internal to (e.g., within) the first pair of primers and
at least one of the
.. nested primers, however, according to the subject invention, needs to be
PKD-specific.
Using the nested PCR procedure, the template that is successfully amplified is
selected
twice for PKD-specificity. The use of nested PCR can also greatly enhance the
yield of the
species-specific product, therefore the sensitivity of the assay, when a
single primer pair fails by
itself.
A sample comprising genomic DNA or cDNA may be used to provide DNA template
for
the amplification reaction. Preferably, genomic DNA is used as template. When
a sample
comprising genomic DNA is used in the reaction mixture, a pair of primers
comprising at least
one selected from the group consisting of SEQ ID NOs. 3-49 generate at least
two specifically
amplified product, one from each PKD allele in the genomic DNA sample.
E. Amplification Controls
Control primers can be used to serve as positive control for the PKD-specific
amplification. The control primers may be added to the same reaction mixture
for PKD-specific
amplification, or it may be added to a control reaction which is run in the
same PCR apparatus
under the same parameters. A control primer may comprise a sequence
complementary to any
identical sequence between a PKD gene and a PKD homologue. Preferably, the
control primers
generate a single amplified product whose size is distinguishable from that
amplified by a pair of
primers comprising at least one PKD-specific primers. The size of the
amplified product by the
control primers may be greater or smaller than the size of the amplified
products generated by
the pair of primers comprising at least one PKD-specific primers. Preferably,
the control primers
are chosen to generate a control product which has at least 100 bp, more
preferably at least 500
bp, more preferably at least 1000 bp difference in size compared to the
amplified product
generated in the same amplification reaction by the pair of primers comprising
at least one PKD-
specific primers.
59
CA 02872385 2014-11-27
A control amplification is especially important when analyzing a PKD allele
with
deletions at the location where a PKD-specific primer anneals. The lack of a
specific
amplification in the presence of an amplified control product may indicate the
presence of the
deletion at a specific location of a PKD gene. In some embodiments, more than
one pair of
control primers is used in the reaction mixture.
See Example 2 for various controls that might be used for the genetic testing
method of
the invention.
Amplified products may be purified to get rid of free primers used in the
amplification by
methods known in the art (e.g., Current Protocols in Molecular Biology,
supra). In a preferred
embodiment, the PCR products are purified using the QuickstepTm 96 well PCR
Purification Kit
from Edge Biosystems.
VII. Detecting the Presence of PCR Amplified Products
The cycle of DNA denaturation, primer annealing and synthesis of the DNA
segment
defined by the 5' ends of the primers is repeated as many times as is
necessary to amplify the
template target until a sufficient amount of either a species-specific or a
universal product is
available for detection. At the conclusion of the amplification reaction, the
presence of amplified
products may be detected using techniques conventional in the art.
The primers may be labeled for facilitating the detection. The primers can be
labeled
with a directly detectable tag, for example a radioactive label such as 32p,
35s, vic or 12si, a
fluorescent compound such as fluorescein or rhodamine derivatives, an enzyme
such as a
peroxidase or alkaline phosphatase, or avidin or biotin. The PKD-specific
primers used to
generate the PKD-specific product and the control primers used only to
generate the control
product may have the same or different labels.
In a preferred embodiment, the amplification products are conveniently
analyzed by gel
electrophoresis.
Electrophoresis is conducted under conditions which effect a desired degree of
resolution
of fragments. A degree of resolution that separates fragments that differ in
size by as little as
=
CA 02872385 2014-11-27
about 500 bp is usually sufficient. Preferably, the resolution is at about 100
bp. More
preferably, the resolution is at about 10 bp. Size markers may also be run on
the gel to permit
estimation of the size of fragments. Preliminary analysis of the size of
specifically amplified
products may indicate insertions or deletions within a P1(0 gene, and the
information obtained
can be interpreted together with results obtained from subsequent DHPLC and
sequence
analysis.
The amplification product pattern may be visualized. Where an amplification
primer has
been labeled, this label may be revealed. A substrate carrying the separated
labeled DNA
fragments is contacted with a reagent which detects the presence of the label.
For example, an
= amplified product generated from a radioactively labeled primer may be
detected by
radioautography. Where the amplification primers are not labeled, the
substrate bearing the PCR
product may be contacted with ethidium bromide and the DNA fragments
visualized under
ultraviolet light.
VIII. Separating PCR Amplified Products
Under the most stringent condition which only allows the annealing of
completely
complementary sequences but not sequences comprising one or more non-
complementary
nucleotides, a PKD-specific primer will only anneal to an authentic PKD gene
template, but not
a PKD homologue. Therefore, under the most stringent condition, a PKD-specific
primer, in
combination with a primer with opposite orientation, being PKD-specific or
not, will only
produce amplified product from an authentic PKD template, but not from a PKD
homologue.
However, during a typical PCR amplification reaction, a PKD-specific may
anneal to a template
comprising an authentic PICD gene and a PKD homologue, especially due to the
temperature
cycling required by a PCR reaction. Therefore, both specifically amplified
products and non-
specifically amplified products may be produced, although the amount of non-
specifically
amplified products may be reduced by the use of at least one P1(i)-specific
primer.
A. Formation of Homoduplex and Heteroduplex
In one embodiment of the invention, a mixture of homoduplexes and
heteroduplexes is
formed prior to the DHPLC analysis. A standard nucleic acid homoduplex (e.g.,
amplified
61
CA 02872385 2014-11-27
product from a normal PKD allele) may be added to the sample and the mixture
is subjected to
denaturation, e.g. by heating the mixture to about 90 C or about 95 C. The
denatured single
stranded nucleic acids formed during the denaturation process are then
annealed by slowly
cooling the mixture to ambient temperature. A new mixture of homoduplexes and
heteroduplexes is formed if the sample contains a mutation. If the sample does
not contain a
mutation, only a homoduplex of the standard nucleic acid will be formed. In
the preferred
embodiment, the standard nucleic acid is the "normal" nucleic acid.
In most cases, a PKD patient individual is heterozygous at the loci comprising
a PKD
gene. That is, the carrier has only one PKD allele and a mutant form and has
the other allele as a
normal form (e.g., wild type). Since most of the PKD mutations result in a
dominant phenotype,
one mutant allele is sufficient to predispose a risk for ADPKD development.
Another
heterozygous situation is when both alleles are mutated but each carries one
or more different
mutations. For a heterozygous PKD patient, a PCR amplification using a primer
pair comprising
at least one PKD-specific primer, including a nested PCR amplification, would
result in at least
two specifically amplified PKD products, one from each allele. The two
specifically amplified
PKD products may or may not be of the same length (e.g., different length if
the mutation on one
allele comprises a deletion or an insertion) and would differ in at least one
nucleotide from each
other.
The amplified products may be denatured and re-annealed with each other to
form
duplexes. When a specifically amplified product from a normal allele or a
specifically amplified
product from a mutant allele anneals to another specifically amplified product
from the same
allele, they will form homoduplex. However, if a specifically amplified
product from a normal
allele anneals to a specifically amplified product from a mutant allele, they
form a heteroduplex.
In rare cases, a mutation is in homozygous form, that is, both alleles in an
individual
.. (e.g., a PKD patient) comprise the same mutations. If a sample is taken
from a homozygous
PKD patient, the PCR amplification will not generate specifically amplified
products which can
form heteroduplex upon denaturing and re-annealing. In some embodiments of the
invention, a
sample comprising a normal (e.g., a wide type) PKD gene is added to the PCR
reaction mixture
so that amplification using a primer pair comprising at least one PKD-specific
primer will
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CA 02872385 2014-11-27
produce specifically amplified products from the normal PKD gene, therefore
ensuring the
formation of a heteroduplex during the denaturation and re-annealing process
following PCR
amplification.
Homoduplexes formed in the denaturation and re-annealing process may also
include
those formed by non-specifically amplified products. If in very rare cases, a
sequence in a
template allele (e.g., a PKD homologue sequence) which give rise to non-
specifically amplified
products also comprises one or more mutation, a heteroduplex may also form.
The heteroduplex
formed between non-specifically amplified products will also be subjected to
further separating
the identification process.
=
B. Separating and Identifying Heteroduplex
The presence of a heteroduplex formed by PKD-specifically amplified products
indicates
the presence of a mutation in a PKD gene. By separating for heteroduplexes,
one can identify
whether a mutant allele present in the sample, e.g., taken from an individual.
This separating
process gets rid of most of the non-specifically amplified products and
specifically amplified
products from normal alleles, therefore improves the efficiency and
specificity of identifying a
mutant allele and a PKD patient.
It is well known in the DNA art that a heteroduplex strand will denature
selectively at the
site of base pair mismatch, creating a "bubble", at a lower temperature than
is necessary to
denature the remainder of the heteroduplex strand, i.e., those portions of the
heteroduplex strand
which contain complimentary base pairs. This phenomenon, generally referred to
as partial
.
denaturation, occurs because the hydrogen bonds between mismatched bases are
weaker than the
hydrogen bonds between complimentary bases. Therefore, less energy is required
to denature
the heteroduplex at the mutation site, hence the lower temperature required to
partially denature
the heteroduplex at the site of base pair mismatch than in the remainder of
the strand.
Since at least one base pair in a heteroduplex is not complimentary, it takes
less energy to
separate the bases at that site compared to its fully complimentary base pair
analog in a
homoduplex. This results in the lower melting temperature of a heteroduplex
compared to a
homoduplex. The local denaturation creates, what is generally called, a
"bubble" at the site of
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base pair mismatch. The bubble distorts the structure of a DNA fragment
compared to a fully
complimentary homoduplex of the same base pair length. This structural
distortion under
partially denaturing conditions has serves as the basis for DHPLC to separate
heteroduplexes and
homoduplexes.
A separation process called "Denaturing HPLC" (DHPLC) has been used to detect
mutations by separating a heteroduplex (resulting from the presence of a
mutation) and a
homoduplex having the same bp length. DHPLC has been applied to mutation
detection (e.g., see
Underhill, et at, 1997, Genome Research 7:996; Liu, et al., 1998, Nucleic Acid
Res., 26;1396).
This separation is based on the fact that a heteroduplex has a lower melting
temperature (Tm)
than a homoduplex. When DHPLC is carried out at a partially denaturing
temperature, i.e., a
temperature sufficient to denature a heteroduplex at the site of base pair
mismatch,
homoduplexes can be separated from heteroduplexes having the same base pair
length
(Hayward-Lester, et al., 1995, Genome Research, 5:494; Underhill, et al.,
1996, Proc. Natl.
Acad. Sci. USA 93:193; Doris, et al., 1997, DHPLC Workshop, Stanford
University).
Thus,
the use of DHPLC was applied to mutation detection (Underhill, et al., 1997,
Genome Research
7:996; Liu, et al., 1998, Nucleic Acid Res., 26:1396). DHPLC can separate
heteroduplexes that
differ by as little as one base pair under certain conditions.
The change in the structure of DNA from an orderly helix to a disordered,
unstacked
structure without base pairs is called the helix-random chain transition, or
melting. Statistical-
mechanical analysis of equilibria representing this change as a function of
temperature for
double-stranded molecules of natural sequence has been presented by Wartell
and Montroll
(1972, Adv. Chem. Phys. 22: 129). The theory assumes that each base pair can
exist in only two
possible states-either stacked, helical, and hydrogen bonded, or disordered.
It permits calculation
of the probability that each individual base pair is either helical or melted
at any temperature,
given only the base sequence and a very small number of empirically calibrated
parameters. The
statistical-mechanical theories take into account the differing intrinsic
stabilities of each base pair
or cluster of neighboring base pairs, the influence of adjacent helical
structure on the probability
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that a neighboring base pair is helical or melted (the coopertivity), and the
restrictions on the
conformational liberty of a disordered region if it is bounded at both ends by
helical regions.
Iteration of the probability calculation at a closely spaced series of
temperature steps and
interpolation permit determination of the midpoint temperature at which each
base pair is at
50/50 equilibrium between the helical and melted states. The MELT program
provides the
midpoint temperature and some other functions. A plot of midpoint temperature
as a function of
position along the molecule is called a melting map. It clearly shows that the
melting of nearby
base pairs is closely coupled over substantial lengths of the molecule despite
their individual
differences in stability. The existence of fairly long regions, 30-300 bp,
termed domains, in
which all bases melt at very nearly the same temperature, is typical. The
melting map directly
delineates the lowest melting domains in the molecules.
At a partially denaturing temperature, a heteroduplex having a base pair
mismatch within
a sample sequence will denature at the site of the mismatch, while the rest of
the sample
sequence will remain intact. The partially denatured heteroduplex can be
separated and detected
using DHPLC.
When HPLC is used under partially denaturing conditions (e.g., DHPLC) to
separate a
mixture of homoduplexes and heteroduplexes, the heteroduplexes usually elute
ahead of the
homoduplexes.
In particular embodiment of the invention, a heteroduplex is separated and
identified
from a homoduplex by DHPLC, and the presence of heteroduplex indicates the
presence of at
least one mutation in the PKD gene, e.g., a substitution of one or more
nucleotides (or insertion
or deletion of one or more nucleotides) present in the mutant allele.
In another particular embodiment, DHPLC gradient is determined by WavemakerTM
4.0
software from Transgenomic, Inc. (San Jose, CA).
Separating applications require that the mutation can be detected regardless
of where the
mutation might be located on the fragment. In this situation, the mutation
might be located in the
middle of the fragment or in a higher melting domain, both cases where it is
more difficult to
CA 02872385 2014-11-27
detect. It is preferred than the range of melting variation of the fragment is
no greater than 10 C
and most preferred is the range of variation is no greater than 5 C.
In some mutation analyses, only two peaks or a partially resolved peak(s) are
observed in
DHPLC analysis. The two homoduplex peaks may appear as one peak or a partially
resolved
peak and the two heteroduplex peaks may appear as one peak or a partially
resolved peak. In
some cases, only a broadening of the initial peak is observed under partially
denaturing
conditions.
If a sample contained homozygous DNA fragments of the same length, then
hybridization and analysis by DHPLC would only produce a single peak at any
temperature since
no heteroduplexes could be formed. In the operation of the present method, the
determination of
a mutation can be made by hybridizing the homozygous sample with the known
wild type
fragment and performing a DHPLC analysis at a partially denaturing
temperature. If the sample
contained only normal allele then a single peak would be seen in the DHPLC
analysis since no
heteroduplexes could be forrned. If the sample contained heterozygous mutant
alleles, then
analysis by DHPLC would show the separation of homoduplexes and
heteroduplexes.
The temperature at which 50% of a constant melting domain is denatured may
also be
determined experimentally by plotting the UV (UV) absorbance of a DNA sample
against
temperature. The absorbance increases with temperature and the resulting plot
is called a
melting profile (Bres'lauer et al., 1986, Proc. Natl. Acad. Sci. USA 83:3746;
Breslauer, 1987,
Calculating Thermodynamic Data for Transitions of any Molecularity, p. 221,
Marky et al. eds.,
J. Wiley and Sons). The midpoint of the absorbance axis on the melting profile
represents the
melting temperature (Tm), i.e. the temperature at which 50% of the DNA strands
in the duplex
are denatured. In one embodiment of the present invention, this observed Tm is
used as a
starting temperature for performing DHPLC for mutation detection. The
temperature may be
then adjusted according to the patterns observed using different controls (see
below). In one
embodiment, a consistent Tm is used to analyze the same amplicons (i.e.,
produced by the same
pair of primers) from different samples.
In another embodiment of the present invention, software such as MELT (Lerman,
et al.,
1987, Meth. Enzymol. 155:482) or WinMeltTm, version 2.0, is used to obtain a
calculated Tm
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CA 02872385 2014-11-27
which is used as a starting temperature for performing DHPLC for mutation
detection. These
software programs show that despite individual differences in base pair
stability, the melting
temperature of nearby base pairs is closely coupled, i.e., there is a
cooperative effect. Thus, there
are long regions of 30 to 300 base pairs, called "domains", in which the
melting temperature is
fairly constant. In a similar manner, the software MELTSCAN (Brossette, et
al., 1994, Nucleic
Acid Res. 22:4321) calculates melting domains in a DNA fragment and their
corresponding
melting temperatures. The concept of a constant temperature melting domain is
important since
it makes possible the detection of a mutation in any portion of the domain at
a single
heteromutant site selective temperature.
10. Another particular method for separating and identifying heteroduplex
is Matched Ion
Nucleic acid Chromatography (MIPC). MIPC was introduced to effectively
separate mixtures of
double stranded nucleic acids, in general and DNA, in particular, wherein the
separations are
based on base pair length (U.S. Patent U.S. Patent Nos. 5,585,236 and
6,287,822; Huber et at.,
1993, Chromatographia 37:653; Huber et al., 1993, Anal. Biochem. 212:351).
mrpC separations
are complete in less than 10 minutes, and frequently in less than 5 minutes.
MIPC systems
(WAVETM DNA Fragment Analysis System, Transgenomic, Inc. San Jose, Calif.) are
equipped
with computer controlled ovens which enclose the columns and column inlet
areas.
Although DHPLC and MICP are the described methods for separating and
identifying
heteroduplex, it is understood that other methods known in the art may also be
used for
identifying heteroduplex. For example, heteroduplex analysis on high
resolution gel matrices are
also able to detect even single nucleotide polymorphisms. (Hauser et at.,
1998, Plant. J. 16:117-
25). The PCR/OLA procedure can be used for analyzing amplification products to
detect SNPs
in the 3' end of the human P1CD gene (Glick and Pasternak, 1994, Molecular
Biotechnology:
Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C.,
pp. 197-200).
Conformation-sensitive gel electrophoresis of amplification products may also
be employed as a
means of analysis by the skilled artisan in practicing the methods of the
present invention.
(Markoff et al., 1998, Eur. J. Genet. 6:145-50). This can also be achieved by
techniques such as
PCR-restriction fragment-SSCP, which can detect single base substitutions,
deletions or
insertions (Tawata et al., 1996, Genet. Anal. 12(3-4):125-27; Lee et at,,
1992, Anal. Biochem.
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CA 02872385 2014-11-27
205:289-93). Electrophoresis for analyzing amplification products is done
rapidly and with high
sensitivity by using any of various methods of conventional slab or capillary
electrophoresis,
with which the practitioner can optionally choose to employ any facilitating
means of nucleic
acid fragment detection, including, but not limited to, radionuclides, UV-
absorbance or laser-
induced fluorescence (Keparnik et al., 1998, Electrophoresis 19:249-55; Inoue
et al. 1998, J.
Chromatogr. A. 802:179-84; Dovichi, 1997, 18:2393-99; Arakawa et al., 1997, J.
Pharm.
Biomed. Anal. 15:1537-44; Baba, 1996, J. Chromatgr B. Biomed. Appl. 687:271-
302; Chan et
al., 1997, J. Chromatog B. Biomed. Sci. Appl. 695:13-15). Any of diverse
fluorescent dyes can
optionally be used to label primers of the present invention or amplification
products for ease of
analysis, including but not limited to, SYBR Green I, Y10-PRO-1, thiazole
orange, Hex (i.e., 6-
carboxy-2',4',7',4,7-hexachlorofluoroscein), pica green, edans, fluorescein,
FAM (i.e., 6-
carboxyfluorescein), or TET (i.e., 4,7,2',7'-tetrachloro-6-carboxyfluoroscein)
(e.g., Skeidsvoll
and Ueland, 1995, Anal. Biochem. 231:359-65; Iwahana et al., 1996,
Biotechniques 21:510-14,
516-19).
In using the invention in its preferred embodiment to effect a separation of
homoduplexes
and heteroduplexes for the purpose of mutation detection, a DNA sample is
hybridized with a
normal DNA fragment by denaturing and annealing the mixture as described
herein above. The
DNA sample can be hybridized with normal DNA directly. The DNA sample can also
be
amplified by PCR and then hybridized with the normal DNA. Alternatively, a
normal fragment
.. may be added to the sample prior to PCR amplification. The amplified
mixture can then be
hybridized following amplification. I n each of these three hybridization
scenarios, a mixture of
homoduplexes and heteroduplexes is produced if a mutation is present in the
sample. The
sample, so prepared, is analyzed by DHPLC under partially denaturing
conditions, preferably at
56 to 58 C, for the presence of a mutation using the method of the invention.
When the method of the invention is used for separating a large number of
samples for
the presence of a mutation, the throughput of samples may be increased
significantly by speeding
up the analysis for each sample using a steeper gradient for the fragment
bracketing range.
In all embodiments and aspects of the invention, the nucleic acid fragments
are detected
as they are separated and eluted from the DHPLC column. Any detector capable
of detecting
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nucleic acids can be used in the DHPLC mutation detection method. The
preferred detector is an
online UV detector. If the DNA fragments are tagged with fluorescent or
radioactive tags, then a
fluorescence detector or radioactivity detector can be employed, respectively.
Following
detection, the separated fragments are displayed on a video display separate
or printed by a
printer. The fragments so displayed appear either as peaks or as bands in a
lane.
C. Quality Controls Helpful for Evaluating DHPLC for PICD-2 and
PKD-1 Unique
Region
The chemical principles which permit DHPLC to distinguish between heteroduplex-
homoduplex mixtures and homoduplexes alone also make the methodology quite
sensitive to (1)
buffer composition, (2) oven temperature at the time of analysis, (3) column
condition, and (4)
system condition at the time a sample is injected. Fluctuation in elution
patterns is normal, and
varies depending on the size and sequence of the amplicon, and the specific
DHPLC conditions
under which it is analyzed. On skilled in the art would have the knowledge in
interpreting the
elution patterns produced, for example, by following the protocol provided by
the manufacture
of the DHPLC equipment. However, limits on the extent of fluctuation are
appropriate to help
ensure that conditions are within a range that would be expected to
effectively separate for DNA
variants. The following quality control requirements are useful examples
established for each
analytical condition to ensure consistent assay performance.
1. No DNA Control
This control demonstrates that reagents and materials are free of non-specific
signal that
could interfere with patient analysis. In some embodiment, the control must
show minimal
signal (<10% of normal control peak height) in a no-DNA sample treated
identically to a sample
comprising a DNA, e.g., extracted from a tissue. Because all of the analytical
system's hardware
is re-used for each sample analysis, and because the DHPLC analysis is the
separating
component, up to 10% peak height of the normal control is permitted. Actual
contamination
with a different sequence might cause a false positive DHPLC pattern
difference which would
trigger reflexing to sequencing which would not be expected to detect a 10%
contaminant. In the
event that a sequence difference is detected, the fragment would be repeated
from the point of
PCR to confirm the result. Similarly contamination of an actual positive with
10% of a normal
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sequence would not be expected to significantly alter the pattern since 50% of
the DNA present
is already normal. Rare cases where a very subtle pattern change might be
obscured by 10%
extra normal DNA in the injection are accounted for in the sensitivity
estimates of 78-96%.
However, persistent no DNA signal each time the amplicon is analyzed indicates
the need to
alter analytical conditions to minimize or eliminate a systematic and
persistent no DNA signal.
2. Normal Control
In one embodiment, the normal control pattern must be consistent with historic
patterns.
Consistency with established patterns indicates acceptable amplification,
retention times, peak
height, and peak shape. Therefore, PCR and DHPLC conditions (machine and
buffers, etc.) are
.. performed as specified in the Examples. Homologues, or other non-specific
amplification
signals are absent as indicated by comparison with the established normal
control pattern.
3. Positive Control
The positive controls are "DHPLC analytical condition controls" used to
demonstrate that
the established DHPLC analytical conditions (which detect the positive control
heteroduplex) are
in effect at the time of analysis. A positive control pattern distinct from
normal control and
consistent with historic patterns indicates acceptable retention time, peak
height(s), peak shape
and pattern. Heteroduplex detection indicates that the specific DHPLC analysis
conditions
optimal for the individual fragment were in effect during patient analysis. It
is important to note
that these controls are not necessarily PKD positive signals. Specific PKD
positive samples for
each of the 83 PKD fragments are not available. In their absence, another
heteroduplex (positive
and normal control) is used as the positive indicator demonstrating
appropriate analytical
conditions at the time of analysis.
4. Additional Positive Controls
Additional positive controls provide pattern(s) consistent with historic
patterns for this
.. specific mutation and may be used to separate out very common
polymorphisms. Generally, a
speeific DNA variant will generate a unique signature heteroduplex pattern
that is highly
reproducible from sample to sample. A pattern consistent with the established
pattern indicates
acceptable retention time, peak height(s), peak shape and pattern. The
specific heteroduplex
CA 02872385 2014-11-27
pattern demonstrates that specific DHPLC analysis conditions optimal for this
DNA variant were
in effect during patient analysis and, therefore, patient patterns matching
this can be considered
to possess the common polymorphism. This optional separating method for common
polymorphisms is highly specific to the unique amplicon and variant and is
dependent upon
appropriate validation studies unique to the variant.
D. Analyzing DHPLC Results
Since DHPLC is a separating process, any specimen (e.g., DNA, or cell lysate
or tissue
sample) with a signal that differs from the normal control should be
considered a potential
positive and treated by one of several options available depending on the
circumstances. For
some embodiments, a signal that is too week to interpret (less than 25% of the
normal control
peak height) could be caused by PCR failure, Wave injection failure, or some
other sporadic
instrumentation problem unique to the sample. Options include repeat from the
point of PCR,
repeat the Wave injection (with all controls), or report the wave result as
inconclusive and
proceed to sequencing. A signal that differs from the normal control in
pattern should be
considered positive, scored as "P", and sequenced. A signal that differs very
slightly from the
normal control pattern should be scored as "B" and sequenced. A signal that is
much stronger
than the normal control signal should be scored as "P" and sequenced. Note
that no patient
specimens will be resulted based on these results alone. The specific options
utilized will vary
with the amplicon and its DHPLC performance history, and the specific
circumstances for the
specimen.
In some embodiments, the only results released from the DHPLC results will be
those
scored as "normal" by Wave analysis. In order to be scored as normal, the
specimen's DHPLC
pattern must be consistent with the normal control by the following QC
criteria: (a) peak number,
(b) peak height, (c) peak pattern, (d) retention time, (e) baseline shape. In
other words, the
pattern for the individual specimen must look like the normal control, within
a reasonable
= expected range of variation. Consult with the validation data reference
patterns if necessary.
The sensitivity of DHPLC separating was assessed by counting patterns that
differ substantially
from the normal control. When a pattern genuinely appears to differ from the
normal control,
there should be no doubt ¨ it is scored as positive and sent on for
sequencing. Only those that
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CA 02872385 2014-11-27
meet the requirements for that specific amplicon and have a pattern consistent
with the normal
control should be scored and released as normal.
Specific numerical criteria used for judging "consistent with" include, but
are not limited
to, (a) number of peaks where a peak represents a local maximum in the signal
intensity, (b) peak
heights, or maximum signal intensities, which are usually between 0.5 and 2.0
times the height
of the normal control, (c) retention time of peaks, which must be +1- 60
seconds compared to the
corresponding normal controls. Peak pattern is judged by relative
correspondence of each slope
change within a peak, and relative intensities and retention times of
individual peaks within a
complex pattern. Baseline patterns are usually smooth and consistent in all
samples. A
relatively low baseline change may represent a heteroduplex that elutes and
perhaps melts at
considerably different retention times from the homoduplex peak(s). The
retention time and
peak height criteria for each amplicon are specified in the attached tables in
the Examples.
In one embodiment, the peak pattern assessment is a combination of (1) the
sample signal
satisfying the same run control criteria as the normal control, and (2) the
sample signal pattern
being consistent with the normal control based on the relative comparison for
that run. Normal
control patterns are expected to vary slightly from run to run, and still be
acceptable, so
individual samples scored as normal are a combination of satisfying (1) the
same run control
criteria as the normal control, (2) the relative control criteria inherent in
the comparison of the
normal control to each patient sample, described above. It seems clear that
subtle changes in the
pattern of the patient sample might be consistent with the absolute run
criteria for the normal
control, yet be clearly distinct using relative comparison of normal and
patient within a run. The
relative comparison within a run always supercedes historic patterns, assuming
the normal
control has passed control criteria and the run is accepted.
IX. Verification of Heteroduplex
Optionally, the identified heteroduplex may be verified by means of digesting
the
amplification products with one or more restriction enzymes. The restriction
enzymes useful for
this purpose are selected by comparing the sequences of authentic PKD genes
and PKD
homologues, or by comparing PKD polymorphisms. Useful restriction enzymes
according to the
invention generate distinguishable fragment profiles for an authentic PKD gene
and a PKD
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homologue. Examples of such restriction enzymes include, but are not limited
to, Pst I, Stu I,
Xma I, Mlu I, Pvu H, BssHII, Fsp I, Msc I, and BM I. Useful restriction
enzymes may also
generate distinguishable fragment profile for a normal PKD gene and a mutant
PKD gene. It is
understood that more restriction enzymes may be identified by simply comparing
the sequence
of a PKD gene and a PKD homologue gene or a normal PKD allele and a mutant PKD
allele. A
restriction enzyme with its recognition site or cleavage site in one sequence
altered so as to
abolish or create a cleavage site but not in the other sequence may be
considered a useful
restriction enzyme for the subject invention. Restriction of nucleic acids is
followed by
separation of the resulting fragments and analysis of fragment length or
differential fragment
migration in denaturing high-performance liquid chromatography (DHPLC) or gel
electrophoresis, as above, including restriction-capillary electrophoresis.
X. Sequencing of HeterOduplexes Identified by DHPLC
Heteroduplex indicating the presence of one or more mutation, identified by
DHPLC,
may be cloned, amplified, andior sequenced. Any known sequencing method known
in the art
can be used to sequence the heteroduplex. In some embodiments, the
heteroduplex identified
was used as template for PCR amplification and amplified products are
sequenced by Sequetech
Corporation (Mountain View, CA). In a preferred embodiment, sequencing is
carried out by
using one of the primers with ID NOs. 3-49.
In some embodiments, the identified heteroduplex is amplified and cloned into
a plasmid
(e.g., Zero Blunt TOPO PCR cloning kit, Invitrogen, Carlsbad, CA, Cat #4560-
01) before
sequencing. The plasmid containing the PCR fragment is then propagated by well
known
methods in the art before subject to sequencing.
XI. DNA Alterations Identified According to The Methods of The Present
Invention
A number of nucleotide and amino acid alterations have been identified in
individuals
diagnosed with ADPKD. Figure 14 summarizes a list of non-limiting examples of
alterations
identified in PKD-1 and PKD-2 nucleotide and amino acid sequences from ADPKD
patients
according to one embodiment of the invention. The sequence positions indicated
in Figure 14
correspond to the nucleotide or amino acid positions as disclosed in Figures
15 and 16, for PKD-
1 and PKD-2 (without introns) respectively.
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The nucleotide and amino acid alterations listed in Figure 14 include both
known
alterations in the art and novel alterations identified the first time by
applicants of the present
invention. Both known and novel alterations are identified in the present
invention as to be
associated with an individual diagnosed with ADPKD, therefore, both known and
novel
alterations disclosed in Figure 14 may be used as markers for diagnosing PICD-
caused ADPKD
or for any other clinical use as described below. Primers which can be used to
identify each
nucleotide sequence alteration are indicated in Figure 14 as well, e.g., as
PK.D1X1, PK.D1X36,
etc. The sequences of the primers are disclosed in Table 3 herein above.
In one embodiment, the invention provides a primer selected from the group
consisting of
10. SEQ ID NOs. 3-49.
In one embodiment, the invention provides an isolated PKD-1 or PKD-2
polynucleotide
comprising one or more nucleotide sequence alterations as disclosed in Figure
14.
In another embodiment, the invention provides an isolated PKD-1 or PKD-2
polynucleotide comprising one or more novel nucleotide sequence alterations as
disclosed in
.. Figure 14 (indicated by bold text).
In another embodiment, the invention provides a purified PKD-1 or PKD-2
polypeptide
comprising one or more amino acid sequence alterations as disclosed in Figure
14.
In another embodiment, the invention provides a purified PKD-1 or PKD-2
polypeptide
comprising one or more novel amino acid sequence alterations as disclosed in
Figure 14
(indicated by bold text).
Preferably, the P1(13-1 or PKD-2 polynucleotide or polypeptide comprising one
or more
sequence alterations is used as a marker for ADPKD.
XII Clinical Use of the Subject Method and Identified Alterations
The genetic testing method described in this application is targeted toward
identifying
DNA alterations in the coding region of the PK.D-1 or PKD-2 gene, including
the splice junction
acceptor/donor sequences, which have been reported to cause ADPKD. The method
can be
performed to assists physicians to:
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CA 02872385 2014-11-27
A. Diagnose PKD-caused ADPKD in symptomatic individuals.
B. Follow up on ultrasound results indicating the presence of one or two
cysts in an
individual at or near the age of onset.
C. Diagnose between different variants of ADPKD (type 1. and 2), which may
or
may not be feasible to determine from family history, ultrasound and other
clinical data.
In one embodiment, the invention provides a method for diagnosing ADPKD in an
individual, comprising identifying nucleotide sequence of PKD-1 or PKD-2 gene
of the
individual, where the existence of one or more nucleotide sequence alterations
in the nucleotide
sequence of PKD-1 or PKD-2 gene as disclosed in Figure 14 is indicative of
ADPKD in the
individual.
Determine and provide genetic counseling for other at-risk family members once
an
ADPKD proband has been identified in a family.
E. Determine the suitability of a living related donor in
transplantation cases.
The invention provides methods for detecting the presence of absence of mutant
PKD
genes, and the presence or absence of ADPKD.
In one embodiment, the present invention provides a method for determining in
an
individual the presence or absence of a mutant PKD gene, comprising the steps
of a) identifying
the nucleotide sequence of a PKD-1 or PKD-2 gene of the individual; b)
comparing the
nucleotide sequence of step a) to the nucleotide sequence alteration in the
nucleotide sequence of
a PKD-1 or PKD-2 gene as disclosed in Figure 14; and c) detecting the presence
of one or more
of the nucleotide sequence alterations disclosed in Figure 14; wherein the
presence of at least one
of the nucleotide sequence alterations is indicative of ADPKD in the
individual; and wherein the
absence of any of said nucleotide sequence alterations indicates the absence
of a mutant PKD-1
and/or PKD-2 gene.
XIII. Kits
CA 02872385 2014-11-27
The invention also provides kits for performing the mutation analysis method
and the
PKD patient identification method of the invention. The invention provides for
kits for detecting
the presence of absence of mutant PKD genes, and the presence or absence of
ADPKD.
Embodiments of the subject kits, in accordance with the methods of the
invention,
include at least one isolated first nucleic acid selected from the group of
SEQ ID NOs. 3-49
and/or their complementary sequences. The kit may further comprise at least
one isolated
second nucleic acid which has an opposite orientation from the first nucleic
acid, and where the
first and second nucleic acids amplify a fragment of a template nucleic acid
comprising a
sequence of SEQ ID NO. 1 or 2, and packaging materials therefore. The kit of
the invention may
further comprises at least one component selected from the group consisting
of: a DNA
polymerase, a template nucleic acid, a restriction enzyme, a control
oligonucleotide primer,
ddNTPs, a PCR reaction buffer and the combination thereof. Kits of the
invention, in addition to
the reagents, preferably include written instructions for performing the
subject methods. Kits are
preferably packaged in a unit container and may contain the reagents in pre-
measured amounts
designed to operate with each other so as to produce the desired result.
Examples
The invention is illustrated by the following non-limiting examples wherein
the following
materials and methods are employed.
Example 1 Reagents, Special Supplies and Equipment
A. Chemicals
The following is a listed of chemicals used for PKD-1 amplification and DHPLC
(WAVE) analysis.
1% Agarose, IX TBE, 54 Well Gel with Ethidium Bromide (Embitec, Catalog Number
GE 4580)
2% Agarose, IX TBE, 54 Well Gel with Ethidium Bromide (Embitec, Catalog Number
GE 4582)
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96 Well Gel Filtration Block (Edge Biosystems, Catalog Number 91751)
Quickstep' 96 Well PCR Purification Kit (Edge Biosystems, Catalog Number
99605)
AmpliTaq Gold with GeneAmp PCR Buffer II & MgC12 Solution (Perkin Elmer,
Catalog
Number N808-0241)
rTth DNA Polymerase, XL & XL Buffer II Pack (Perkin Elmer, Catalog Number N808-
00193)
TapPlus Precision PCR System (Stratagene, Catalog Number 600211)
Dimethyl Sulphoxide (DMSO) (Sigma, Catalog Number D-2650)
Ready-Load 100 bp DNA Ladder or Equivalent (Gibco BRL, Catlaog Number 10380-
012)
Ready-Load 1 kb DNA Ladder or Equivalent (Gibco BRL, 1800-828-6686, Catlaog
Number 10381-010)
Big Dye Terminator Ready Reaction Kit (Perkin Elmer, Catalog Number 4303150)
Gel Filtration Cartridge.(Edge Biosystems, Catalog Number 42453)
Long Ranger Singeem packs (FMC BioProducts, Catalog Number 50691 or 50693).
Oligonucleotides (Operon Technologies, Inc.)
WAVE Mutation Standard (209 bp), Catalog Number 560077 (180 ul)
Acetonitrile-HPLC Grade (VWR, Catalog Number BJ015-1)
HPLC Grade Water (VWR, Catalog Number BJ365-4)
Triethylammonium Acetate (TEAA) (Transgenomic, Catalog Number SP5890)
B. Reagents and Solutions
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1.1.N1 oligonucleotide primers: 10p.M working aliquots of PCR primers
dissolved in TE
buffer should be stored at 4 C in Pre-PCR refrigerator; sequencing primer
working aliquots
should be stored at 4 C in Post-PCR refrigerator.
Solution X-127: Upgrade Blue Dextran in 50 mM EDTA (01=8.0)
5 Combine 0.5 ml 50 mM EDTA pH=8.0 (Solution X-35), 500 mg Blue Dextran AND
9.5
ml AUTOCLAVED, STERILE FILTERED DiH20 in a sterile 15 ml conical centrifuge
tube.
Thoroughly mix the solution by vortexing.
Solution X-126: Upgrade Gel Loading Buffer: Combine 200p1 deionized Formarnide
and 40 il Upgrade Blue Dextran in 50 mM EDTA (Solution X-127) in a 1.5 ml
sterile
10 .. microcentrifuge tube. Vortex thoroughly.
WAVE Solution A: Solution A (0.025% ACN)
Preparation of 2L: 100m1 Ion Pairing Agent (TEAA)
500ulAcetonitrile (ACN)
Top to 2L with HPLC grade water
WAVE Solution B: Solution B (25% ACN)
Preparation of 2L: 100m1 Ion Pairing Agent (TEAA)
500m1 Acetonitrile (ACN)
Top to 2L with HPLC grade water
WAVE Syringe Wash Solution: _Syringe Wash (8% ACN)
Preparation of 2L: 160m1 Acetonitrile (ACN)
Top to 2L with HPLC grade water
WAVE Solution D: Solution D (75% ACN)
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Preparation of 2L:500m1HPLC grade water
Top to 2L with Acetonitrile (ACN)
C. Equipment and Special Supplies
Table 5
Perkin Elmer ABI PrismTM 377 DNA Sequencer
761 Main Avenue
Norwalk, CT 06859
VWR Scientific Products 1. Beckman AllegraTM 21 Centrifuge
P.O. Box 232 2. Eppendorf Microcentrifuge 5415C
=
Boston, MA 02101 3. Multichannel pipet
4. Sterile reservoirs
5. DURX 670 wipers
6. VWR Model 1300U Oven
Transgenomic, Inc. WAVE Nucleic Acid Fragment Analysis
System
12325 Emmet Street
Omaha, NE 68164
Example 2. Procedure
Stage I: Preparation Of DNA and /or RNA From Patient Specimens =
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CA 02872385 2014-11-27
DNA is extracted from whole blood or lymphocytes using the Puregene DNA
extraction kit. DNA extracted using these reagents should be successfully PCR
amplified under
the conditions specific to the assay. This is tested by performing the assay
as specified in the
protocol and comparing the results obtained with the positive DNA control that
has been
previously validated.
Extracted DNA is quantitated and the 260/280 ratio is 1.4 or greater. Samples
with lower
ratios indicate that the quality of DNA is poor and may not meet PCR
standards. If end results of
the assay are not interpretable the sample should be re-extracted.
Stage II: Amplification of DNA by PCR
PCR reaction mixtures and cycling parameters (e.g., for exon 1 of PKID-1 gene)
were set
up as illustrated in Table 5. PCR conditions were set up similarly, but
optimized for specific and
efficient amplification of other exons.
Table 6. PCR Reaction Master Mix Component Concentrations and Thermal
Cycling Conditions For First round PCR Products 1-8 (L1-1,8)
LOWER MASTER Mix:
Component: Reaction Volume/
Concentration reaction
Water 13.0u1
10X Buffer 1X 2.0 ul
Mg(Oac)2 0.9 mM None
dNTP mix 200 uM 1.0 ul
Primer 1 0.25 uM 1.25 ul
CA 02872385 2014-11-27
Primer 2 0.25 uM 1.25 ul
DMSO 7.5% 1.5 ul
TOTAL VOLUME 20 ul
One wax bead was added to each well and incubated in a therrnal cycler @ 80 C
for 5
minutes to melt the wax and incubated at 25 C for an additional 5 minutes
before placed on ice
for further handling.
UPPER MASTER MIX:
Component: Reaction Volume/
Concentration reaction
Water 23.15u1
10X Buffer 1X 3.0 ul
TaqPlus Precision
Polymerase mixture 5U/rxn 1.0 ul
DMSO ' 7.5% 2.25 ul
TOTAL VOLUME 29.4u1
Genomic DNA @ 500 ng/ul 0.6 ul
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Cycling Parameters
Melting the Wax Amplification
80 C 5 min 1 cycle 94 C 3 min 1 cycle
25 C forever
96 C 30 sec
*Add Upper Master Mix and DNA before 68 C 20 sec 35 cycles
proceeding to next cycling step. 72 C 3 min + 4sec/cyc1e
72 C 19 min 1 cycle
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Table 7. Example of nested PCR reaction setup
REAGENT STOCK VOLUME PER REACTION
CONCENTRATION REACTION CONCENTRATION
Water 31.O pi
Buffer II 10X 5.0 ul lx
MgC12 25 mM 2.0 ul 1.0 mM
DNTP mix 10 mM each 1.0 ul 200 AM each
CAD-18-PF1 (primer) 10 uM 3.0 ul 0.6 AM
CAD-18-PR1 10 p.M 3.0 pl 0.6 p.M
(primer)
DMSO 100% 2.5 ul 5%
Amplitaq Gold 5U/pi O.5 pi 2.5U
TOTAL 48.0 ,1
Table 8. Summary of Amplification Conditions For Exons
CYCLE NUMBER TEMPERATURE TIME DESCRIPTION
1 cycle 94 C 10 min AmpliTaq Gold activation
92 C 1 min Denaturing
35 cycles 55 C 1 min Annealing
72 C 1 mm Extension
83
=
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1 cycle 72 C 10 min Final extension
(hold) 4'C forever
PCR amplified fragments may be compared in size, signal intensity and
migration pattern
with positive control DNA control that has been previously validated. The size
of the PCR
amplified fragments is determined by comparison to the Molecular weight marker
(DNA
MASSTM Ladder-Gibco BRL) on the gel. The low range DNA Mass Ladder gives 6
bands of
double stranded (100-2000 bp) DNA on staining the gel with ethidium bromide.
Stage III: DHPLC Analysis of PCR Products
Heteroduplexes formed by PCR amplified products are analyzed using WAVE
nucleic
acid fragment analysis system from Transgenomic, Inc. (Omaha, NE 68164).
Stage IV: Cycle Sequencing
Tables 9 and 10 provide examples of sequencing conditions used in one
embodiment of
the invention.
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CA 02872385 2014-11-27
Table 9. Sequencing Reaction Master Mix Component
REAGENT STOCK VOLUME PER REACTION
CONCENTRATION REACTION CONCENTRATION
Water 14.0 ul
Big Dye Terminator 2.5X 4.0 ul 0.5X
Ready Reaction Mix
Primer = 10 laM 1.0 ul 0.5 uM
FINAL VOLUME 19.0 ul
Table 10. Cycle Sequencing Conditions
CYCLE NUMBER TEMPERATURE TIME DESCRIPTION
94 C 10 sec Denaturing
30 cycles 55 C 5 sec Annealing
60 C 4 min Extension
(hold) 4 C forever
Example 3. Summary of Results
In one experiment, detection of mutations in exons 1-34 of the PKD-1 gene was
achieved
by using eight sets of oligonucleotide primers in eight separate first round
PCR reaction to
amplify DNA fragments of the following sizes: a) LR1 was 2.2 kb and contains
exon 1. b) LR2
was 4.6 kb and contains exons 2-7. c) LR3 was 4.2 kb and contains exons 8-12.
d) LR4 was 4.4
kb and contains exons 13-15. e) LR5 was 3.4 kb and contains exons 15 (3'-end)
through 21. 0
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LR6 was 0.3 kb and consists of exon 22. g) LR7 was 4.2 kb and contains exons
23-28. II) LR8
was 5.8 kb and contained exons 29-34 of the duplicated region of the gene. The
amplified
product from the first round of amplification were then serially diluted to
1:104 or 1:105 to
remove genomic contamination and subsequently used as template in a second
round of nested
PCR. The nested PCR products were heteroduplexed and screened for sequence
alterations by
DHPLC. Each fragment was analyzed against a normal and positive control using
a temperature
and acetonitrile gradient specific to the amplicon. Any samples testing
positive by DHPLC
analysis were subsequently purified and sequenced. Cycle sequenced products
were then
separated on an ABI 377 automated sequencer and the results were analyzed
using an assortment
of sequencing software. Tables 11-12 and figures Ito 13 illustrate the results
and procedures of
some embodiments of the invention.
Table 11. Numbers of products
analyzed for each PKD gene
Analysis: PICD-1 PKD-2 Total
First Round PCRs 8 8
Amplicons 66 17 83
DHPLC analyses 133 33 166
Base Pairs evaluated 13,830 3204 17,034
Table 12. Variant detection rates
Source of Naturally occurring - Naturally
occurring - Mutagenesis Gene Total
Variant
Independent SSCP Sequence confirmed
Sequence confirmed Separated
PKD-1 14/18 15/17 45/47 74/82
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78% 88% 96% ___________ 90%
PKD-2 20/21 0/0 22/23 42/44
95% 96% 95%
Type 34/39 15/17 67/70 116/126
total
87% 88% 96% 92%
Other Embodiments
The foregoing examples demonstrate experiments performed and contemplated by
the
present inventors in making and carrying out the invention. It is believed
that these examples
include a disclosure of techniques which serve to both apprise the art of the
practice of the
invention and to demonstrate its usefulness. It will be appreciated by those
of skill in the art that
the techniques and embodiments disclosed herein are preferred embodiments only
and that in
general numerous equivalent methods and techniques may be employed to achieve
the same
result.
87
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