Note: Descriptions are shown in the official language in which they were submitted.
CA 02219933 1997-10-31
WO 96/41002 PCTAUS96/08806
~I~SFORTHEnD~NTn~CAl~ON OFGENE~C MODn~CAnON OFDNAnNVOLVnNG DNA
SEQUENCnNG AND POS~ONAL CLONnNG
Field of the Invention
..,
This invention pertains to high-throughput
methodology that directly identifies previously
unidentified sequence alterations in DNA, including
specific disease-causing DNA sequences in m~mm~ , The
methods of the present invention can be used to identify
genetic polymorphisms, to determine the molecular basis for
genetic diseases, and to provide carrier and prenatal
~ nosis for genetic counseling.
Background of the Invention
The ability to detect alterations in DNA
sequences (e.g. mutations and polymorphisms) is central to
the diagnosis of genetic diseases and to the identification
of clinically significant variants of disease-causing
microorg~n; ~m~ . One method for the molecular analysis of
genetic variation involves the detection of restriction
fragment length polymorphisms (RFLPs) using the Southern
blotting technique (Southern, E.M., J. Mol. Biol., 98:503-
517, 1975. Since this approach is relatively cumbersome,
new methods have been developed, some of which are based on
the polymerase chain reaction (PCR). These include: RFLP
analysis using PCR (Chehab et al., Nature, 329:293-294,
1987; Rommens et al., Am. J. Hum. Genet., 46:395-396,
1990), the creation of artificial RFLPs using primer-
specified restriction-site modification (Haliassos et al.,
Nuc. Acids Res., 17:3606, 1989), allele-specific
amplification (ASA) (Newton CR et al., Nuc. Acids Res.,
17:2503-2516, 1989), oligonucleotide ligation assay (OLA)
(T~n~gren U et al., Scie~ce 241:1077-1080, 1988), primer
extension (Sokolov BP, Nuc. Acids Res., 18:3671, 1989),
artificial introduction of restriction sites (AIRS) (Cohen
LB et al., Nature 334:119-121, 1988), allele-specific
CA 02219933 1997-10-31
W O 96/41002 PCTrUS96/08806
oligonucleotide hybridization (ASO) (Wallace RB et al.,
Nuc. Acids Res., 9:879-895, 1981) and their variants.
Together with robotics, these techniques ~or direct
mutation and analysis have helped in reducing cost and
increasing throughput when only a limited number of
mutations need to be analyzed for e~icient ~;~gnostic
analysis.
These methods are, however, limited in their
applicability to complex mutational analysis. For example,
in cystic ~ibrosis, a recessive disorder affecting 1 in
2000-2500 live births in the United States, more than 225
presumed disease-causing mutations have been identified.
Furthermore, multiple mutations may be present in a single
affected individual, and may be spaced within a few base
pairs of each other. These ph~nnm~n~ present unique
difficulties in designing clinical screening methods that
can accommodate large numbers of sample DNAs.
Shuber et al., Hum. Mol . Gen., 2:153-158, 1993,
disclose a method that allows the simultaneous
hybridization o~ multiple oligonucleotide probes to a
single target DNA sample. By including in the
hybridization reaction an agent that ~l;m;n~tes the
disparities in melting temperatures of hybrids formed
between synthetic oligonucleotides and target DNA, it is
possible in a single test to screen a DNA sample ~or the
presence of di~erent mutations. Typically, more than 100
ASOs can be pooled and hybridized to target DNA; in a
second step, ASOs from a pool giving a positive result are
individually hybridized to the same DNA. Shuber et al.,
Genome Res . 5:488-93, 1995, disclose a method for multiple
allele-speci~ic disease analysis in which multiple ASOs are
first hybridized to a target DNA, followed by elution and
se~uencing of ASOs that hybridize. This method allows the
identification of a mutation without the need ~or many
CA 02219933 1997-10-31
WO 96/41002 PCTrUS96/08806
individual hybridizations involving single ASOs and
requires prior knowledge of relevant mutations.
To achieve adequate detection frequencies for
rare mutations using the above methods, however, large
numbers of mutations must be screened. To identify
previously unknown mutations within a gene, other
methodologies have been developed, including: single-
strand conformational polymorph; ~m.s (SSCP) (Orita M et al.,
Proc. Natl. Acad. Sci USA 86:2766-2770, 1989), denaturing
gradient gel electrophoresis (DGGE) (Meyers RM et al.,
Nature 313:495-498, 1985), heteroduplex analysis (HET)
(Keen j. et al., Trends Genet. 7:5, 1991), chemical
cleavage analysis (CCM) (Cotton RGH et al., Proc. Natl.
Acad. Sci. USA 85:4397-4401, 1988), and complete sequencing
of the target sample (Maxam AM et al., Methods Enzymol.
65:499-560, 1980, Sanger F. et al., Proc. Natl. Acad. Sci.
USA 74:5463-5467, 1977). A11 of these procedures however,
with the exception of direct sequencing, are merely
screening methodologies. That is, they merely indicate
that a mutation exists, but cannot specify the exact
sequence and location of the mutation. Therefore,
identification of the mutation ultimately requires complete
sequencing of the DNA sample. For this reason, these
methods are incompatible with high-throughput and low-cost
routine diagnostic methods.
Thus, there is a need in the art for a relatively
low cost method that allows the efficient analysis of large
numbers of DNA samples for the presence of previously
unidentified mutations or sequence alterations.
Summary of the Invention
~35 The present invention encompasses high-throughput
methods for identifying one or more genetic alterations in
CA 02219933 1997-10-31
W O 96/41002 PCT~US96/08806
a target sequence present in a first DNA sample. The
method is carried out by the steps of:
a) hybridizing the first sample with a second DNA
sample not cont~;n;ng genetic alterations to form
heteroduplex DNA cont~;n;ng a mismatch region at the site
of a gehetic alteration(s);
b) cleaving one DNA strand of the heteroduplex in
the target sequence to form a single-stranded gap across
the site of the alteration;
c) treating the cleaved heteroduplex with a DNA
polymerase in the presence of dideoxynucleotides to
determine the seguence across the gap; and
d) comparing the nucleotide sequence across the
gap with a predetermined cognate wild-type sequence to
identify the genetic alteration(s).
In practicing the above-described methods, the
first DNA sample contA;n;ng the target sequence is
hybridized under stringent conditions with a second DNA
sample not cont~;n;ng the alteration. The hybrids that
form contain mismatch regions, which are recognized and
~n~onllcleolytically cleaved on one or both sides of the
mismatch region by mismatch recognition protein-based
systems. When a single endonucleolytic cleavage occurs on
only one side of the mismatch region, one or more
exonucleases are used to form the single-stranded gap.
When ~n~nl~cleolytic cleavage occurs on both sides of the
mismatch region, the single-stranded fragment is released
by the action of a helicase to form the single-stranded
gap. Det~rm;n~tion of the sequence across the gap is
achieved in a single step by an enzymatic DNA sequencing
reaction using dideoxynucleotides and DNA polymerase I, DNA
polymerase III, T4 DNA polymerase, or T7 DNA polymerase.
In an alternate embodiment, the present invention
encompasses high-throughput methods for identifying one or
more genetic alterations in a target sequence present in a
CA 02219933 1997-10-31
W O 96/41002 PCT~US96/08806
first DNA sample. This method is carried out by the
steps of:
a) hybridizing the ~irst sample with a second DNA
sample not cont~;n;ng genetic alterations to ~orm
heteroduplex DNA having free ends and contA;n;ng a mismatch
region at the site of a genetic alteration(s);
b) cleaving the DNA at or in the vicinity of the
alteration, forming new ends;
c) ligating an oligonucleotide of predetermined
sequence to the new ends;
d) det~rm;n;ng the nucleotide sequence adjacent
to the ligated oligonucleotide; and
e)comparing the nucleotide sequence determ;ne~ in
d) with a predetermined cognate wild-type sequence to
identify the genetic alteration(s).
Specific cleavage at or near the alteration is
achieved by hybridizing the first DNA sample cont~;n;ng the
target sequence with a second DNA sample not cont~;n;ng the
alteration, so that heteroduplexes are formed that contain
mismatch regions, which can be recognized and cleaved by
mismatch recognition systems.
Typically, the ~irst DNA sample comprises genomic
DNA from a patient suffering from a genetic disease whose
genome does not contain any o~ the known mutations that
cause that disease, and the target sequence comprises a
known disease-causing gene. The genetic alterations
identified by these methods include additions, deletions,
or substitutions of one or more nucleotides.
Mismatch recognition, cleavage, and excision
systems useful in practicing the invention include without
limitation bacteriophage resolvases, mismatch repair
proteins, nucleotide excision repair proteins, chemical
modification of mismatched bases followed by excision
repair proteins, chemical modification and cleavage, and
CA 02219933 1997-10-31
W O 96/41002 PC~r~US96/08806
combinations thereof, with or without supplementation with
exonucleases as required.
The present invention finds application in high-
throughput methods for multiplex identification of new
mutations or previously unidentified polymorph;.cmc, in
which DNAs obtained from a multiplicity of patients are
immobilized on a single solid support, followed by one or
more of the following steps: hybridization, mismatch
recognition, excision, cleavage, ligation, sequencing, and
sequence comparison steps as set forth above. Furth~rm~re,
multiple specific target sequences can be analyzed
simultaneously by amplifying the target sequences prior to
immobilization, followed by the steps as set forth above.
In an alternate embodiment, the present invention
provides methods for positional cloning of a disease-
causing gene. Invention methods are carried out using the
following steps:
a) hybridizing a first DNA sample derived from an
individual suffering from the disease with a second DNA
sample derived from a multiplicity of individuals not
suffering from the disease, to form hybrids cont~;n;ng
mismatch regions at sites at which the sequence of the
first DNA sample diverges from the sequence of the second
DNA sample;
b) cleaving one DNA strand in the hybrids to form
a single-stranded gap across the site of the alteration;
c) det~rm;n;ng the nucleotide sequence across the
gap;
d) preparing a synthetic oligonucleotide
comprising all or part of the nucleotide sequence
determined in c)i and
e) identifying a DNA clone derived from a cosmid
or a Pl library cont~;n;ng the sequence of the synthetic
oligonucleotide prepared in d).
CA 02219933 1997-10-31
W O 96/41002 PCT~US96/08806
In practicing the present invention, mismatch
regions are recognized and endonucleolytically cleaved on
one or both sides of the mismatch region by mismatch
recognition protein-based systems. When a single
~n~7.o~77CleolytiC cleavage occurs on only one side of the
mismatch region, one or more exonucleases are used to form
the single-stranded gap. When endonucleolytic cleavage
occurs on both sides of the mismatch region, the single-
stranded fragment is released by the action of a helicase
to form the single-stranded gap. Det~rm;n~tion of the
sequence across the gap is achieved in a single step by an
enzymatic DNA sequencing reaction using dideoxynucleotides
and DNA polymerase I, DNA polymerase III, T4 DNA
polymerase, or T7 DNA polymerase.
The present invention further provides alternative
methods for positional cloning of a gene of interest.
These methods are carried out by:
a) hybridizing a first DNA sample derived from an
individual displaying a given phenotype with a second DNA
sample derived from one or more individuals not displaying
the phenotype, to form heteroduplex DNA having free ends
and cont~;n;ng a mismatch region at sites at which the
sequence of the first DNA sample diverges from the sequence
of the second DNA sample;
b) blocking the free ends on the hybrids formed
in a);
c) cleaving one or both DNA strands within or
adjacent to the mismatch regions to form new ends;
d) ligating a single-stranded oligonucleotide of
predetermined sequence to the new ends formed in c);
e) det~rm;n;ng the nucleotide sequence adjacent
- to the ligatedpredetermined sequence;
f) preparing a synthetic oligonucleotide
~35 comprising all or part of the nucleotide sequence
determined in e); and
CA 02219933 1997-10-31
W O 96/41002 PCTrUS96/08806
g) identifying a DNA clone derived from a cosmid
or a Pl library cont~;n;ng the sequence of the synthetic
oligonucleotide prepared in ~).
As used herein, positional cloning refers to a
process by which a previously unknown disease-causing gene
is localized and identified.
The genetic alterations identified by invention
methods include additions, deletions, or substitutions of
one or more nucleotides. Mismatch recognition, cleavage,
and excision systems useful in practicing the invention
include without limitation mismatch repair proteins,
nucleotide excision repair proteins, bacteriophage
resolvases, chemical modification of mismatched bases
~ollowed by excision repair proteins, and combinations
thereo~, with or without supplementation with exonucleases
as required.
Detailed Description of the Invention
The present invention encompasses high-throughput
methods for identifying specific target sequences in DNA
isolated from a patient. As used herein, the term high-
throughput refers to a system ~or rapidly assaying large
numbers o~ DNA samples at the same time. The methods are
applicable when one or more genes or genetic loci are
targets of interest. The specific sequences typically
contain one or more sequence alterations relative to wild-
type DNA, including additions, deletions, or substitutions
of one or more nucleotides.
In practicing the methods of the present
invention, the first DNA sample cont~;n;ng the target
sequence is hybridized with a second sample of DNA (or a
pool of DNA samples) cont~;n;ng one or more wild-type
versions of the targeted gene. The methods of the present
CA 02219933 1997-10-31
W O 96/41002 PCT~US96108806
invention take advantage of the physico-chemical properties
of DNA hybrids between almost-identical (but not completely
identical) DNA strands (i.e., heteroduplexes). When a
seguence alteration is present, the heteroduplexes contain
a mismatch region that is embedded in an otherwise
perfectly matched hybrid. According to the present
invention, mismatch regions are formed under controlled
conditions and are chemically and/or enzymatically
modified; the sequences adjacent to, and including, the
mismatch are then determined. Dep~n~; ng upon the mismatch
recognition method used, the mismatch region may comprise
any number of bases, preferably from 1 to about 1000 bases.
The methods of the invention can be employed to
identify specific disease-causing mutations in individual
patients (when the gene or genes responsible for the
disease are known) or previously unidentified polymorphisms
and for positional cloning to identify new genes.
In a preferred embodiment, the specific DNA
sequence comprises a portion of a particular gene or
genetic locus in the patient's genomic DNA known to be
involved in a pathological condition or syndrome. Non-
limiting examples of genetic syndromes include cystic
fibrosis, sickle-cell anemia, t~lAcsemias~ Gaucher~s
disease, adenosine d~Am;nA~e deficiency, alphal-antitrypsin
deficiency, Duchenne muscular dystrophy, familial
hypercholesterolemia, fragile X syndrome, glucose-6-
phosphate dehydrogenase deficiency, hemophilia A,
Huntington disease, myotonic dystrophy, neurofibromatosis
type 1, osteogenesis imperfecta, phenylketonuria,
retinoblastoma, Tay-Sachs disease, and Wilms tumor
(Thompson and Thompson, Genetics in Medicine, 5th Ed. ) .
.35 In another embodiment, the specific DNA sequence
comprises part of a particular gene or genetic locus that
may not be known to be linked to a particular disease, but
CA 02219933 1997-10-31
W O 96/41002 PCT~US96/08806
in which polymorphism is known or suspected. For example,
obesity may be linked with variations in the apolipoprotein
B gene, hypertension may be due to genetic variations in
sodium or other transport systems, aortic aneurysms may be
linked to variations in a-haptoglobin and cholesterol ester
transfer protein, and alcoholism may be related to variant
forms of alcohol dehydrogenase and mitocho~ial aldehyde
dehydrogenase. Furthermore, an individual's response to
medicaments may be affected by variations in drug
modification systems such as cytochrome P450s, and
susceptibility to particular infectious diseases may also
be influenced by genetic status. Finally, the methods of
the present invention can be applied to HLA analysis for
identity testing.
In yet another em~bodiment, the specific DNA
sequence comprises part of a foreign genetic sequence e.g.
the genome of an invading microorganism. Non-limiting
examples include bacteria and their phages, viruses, fungi,
protozoa, and the like. The present methods are
particularly applicable when it is desired to distinguish
between different variants or strains of a microorganism in
order to choose appropriate therapeutic interventions.
1. PREPARATION OF HETERODUPLEXES
.
In accordance with the present invention, the
target sequence is contained within a sample of DNA
isolated from an ~n; m~l or human patient. This DNA may be
obtained from any cell source or body fluid. Non-limiting
examples of cell sources available in clinical practice
include blood cells, buccal cells, cervicovaginal cells,
epithelial cells from urine, ~etal cells, or any cells
present in tissue obtained by biopsy. Body fluids include
blood, urine, cerebrospinal fluid, and tissue exudates at
the site of infection or inflammation. DNA is extracted
CA 02219933 1997-10-31
W O 96/41002 PCT~US96/08806
from the cell source or body fluid using any of the
numerous methods that are st~n~d in the art. It will be
understood that the particular method used to extract DNA
will depend on the nature of the source. The preferred
amount of DNA to be extracted for analysis of human genomic
DNA is at least 5 pg (corresponding to about 1 cell
equivalent of a genome size of 4 x 109 base pairs). In
some applications, such as, for example, detection of
sequence alterations in the genome of a microorganism,
variable amounts of DNA may be extracted.
Once extracted, the sample DNA cont~;n;ng the
target sequence may be employed in the present invention
without further manipulation. Preferably, one or more
specific regions present in the sample DNA may be
amplified. In this case, the amplified regions are
specified by the choice of particular flanking sequences
for use as primers. Amplification at this step provides
the advantage of increasing the concentration of specific
sequences within the sample DNA population. The length of
DNA sequence that can be amplified ranges from 80 bp to up
to 30 kbp (Saiki et al., 1988, Science, 239:487).
Furthermore, the use of amplification primers that are
modified by, e.g., biotinylation, allows the selective
incorporation of the modification into the amplified DNA.
In one embodiment, the first DNA cont~;n;ng the
target sequence, with or without prior amplification of
particular sequences, is bound to a solid-phase matrix.
This allows the simultaneous processing and scr~n;ng of a
large number of patient or first DNA samples. Non-
limiting examples of matrices suitable for use in the
present invention include nitrocellulose or nylon filters,
glass beads, magnetic beads coated with agents for affinity
capture, treated or untreated microtiter plates, and the
like. It will be understood by a skilled practitioner
that the method by which the DNA is bound to the matrix
CA 02219933 1997-10-31
W O 96/41002 PCTAUS96/08806
will depend on the particular matrix used. For example,
b; n~; ng to nitrocellulose can be achieved by simple
adsorption of DNA to the filter, followed by baking the
filter at 75-80~C under vacuum ~or 15 min. to 2h.
Alternatively, charged nylon membranes can be used that do
not require any ~urther treatment of the bound DNA. Beads
and microtiter plates that are coated with avidin can be
used to bind DNA that has had biotin attached (via e.g. the
use of biotin-conjugated primers). In addition, antibodies
can be used to attach DNA to any of the above solid
supports by coating the surfaces with the antibodies and
incorporating an antibody-specific hapten into the DNA. In
a preferred embodiment, DNA that has been amplified using
biotinylated primers is bound to streptavidin-coated beads
(Dynal, Inc., Milwaukee, WI).
In practicing the present invention, the
untreated or amplified ~irst DNA, preferably bound to a
solid-phase matrix, is hybridized with a second DNA sample
under conditions that favor the formation of mismatch
loops. The second DNA sample preferably comprises one or
more "wild-type" version(s) of the target sequence. As
used herein, a ~wild-type~ version of a gene is one
prevalent in the general population that is not associated
with disease (or with any discernable phenotype) and is
thus carried by "normal" individuals. In the general
population, wild-type genes may include multiple prevalent
versions, which contain alterations in sequence relative to
each other that cause no discernable pathological effect;
these variations are designated "polymorphisms" or "allelic
variants~. Most preferably, a mixture o~ DNAs from
"normal~ individuals is used for the second DNA sample,
thus providing a mixture o~ the most common polymorph; ~m.~,
This insures that, statistically, hybrids formed between
the first and second DNA sample will be perfectly matched
except in the region of the mutation, where discrete
mismatch regions will form. In some applications, it is
CA 02219933 1997-10-31
W O 96/41002 PCTrUS96/08806
desired to detect polymorphisms; in these cases,
appropriate sources ~or the second DNA sample will be
selected accordingly. Dep~n~;ng upon what method is used
subsequently to detect mismatches, the wild-type DNA may
also be chemically or enzymatically modified, e.g., to
remove or add methyl groups.
.
Hybridization reactions according to the present
invention are performed in solutions ranging from about 10
mM NaCl to about 600 mM NaCl, at temperatures ranging from
about 37~C to about 65~C. It will be understood that the
stringency of a hybridization reaction is det~rm; n~ by
both the salt concentration and the temperature; thus, a
hybridization performed in 10 mM salt at 37~C may be of
similar stringency to one performed in 500 mM salt at 65~C.
For the purposes of the present invention, any
hybridization conditions may be used that form perfect
hybrids between precisely complementary sequences and
mismatch loops between non-complementary sequences in the
same molecules. Preferably, hybridizations are performed
in 600 mM NaCl at 65~C. Following the hybridization step,
DNA molecules that have not hybridized to the first DNA
sample are removed by washing under stringent conditions,
e.g., O.lX SSC at 65~C.
The hybrids formed by the hybridization reaction
may then be treated to block any free ends so that they
cannot serve as substrates for further enzymatic
modification such as, e.g., by RNA ligase. Suitable
blocking methods include without limitation r~LILovdl of 5
phosphate groups, homopolymeric tailing of 3' ends with
dideoxynucleotides, and ligation of modified double-
stranded oligonucleotides to the ends of the duplex.
CA 02219933 1997-10-31
W O 96/41002 PCT~US96/08806
2. MISMATCH RECOGNITION AND CLEAVAGE
In the next step, the hybrids are treated so that
one or both DNA strands are cleaved within, or in the
vicinity of, the mismatch region. Dep~n~; ng on the method
used for mismatch recognition and cleavage (see below),
cleavage may occur at some predet~rm;ne~ distance from
either boundary of the mismatch region, and may occur on
the wild-type or mutant strand. The "vicinity" of the
mismatch as used herein thus encompasses from 1 to 2000
bases from the borders of the mismatch. Non-limiting
examples of mismatch recognition and cleavage systems
suitable for use in the present invention include mismatch
repair proteins, nucleotide excision repair proteins,
bacteriophage resolvases, chemical modification, and
combinations thereof. These embodiments are described
below.
In general, the mismatch recognition and/or
modification proteins necessary for each embodiment
described below are isolated using methods that are well
known to those skilled in the art. Preferably, when the
sequence of a protein is known, the protein-coding region
of the relevant gene is isolated ~rom the source organism
by subjecting genomic DNA of the organism to amplification
using appropriate primers. The isolated protein-coding DNA
sequence is cloned into commercially available expression
vectors that, e.g., insert an amino acid "purification tag"
at either the amino- or carboxyterminus of the recombinant
protein. The recombinant expression vector is then
introduced into an appropriate host cell (e.g., E. coli),
and the protein is recovered from the cell lysate by
affinity chromatography that recognizes the "tag". For
example, the bacterial expression vector pQiexl2 is used to
express proteins with a polyhistidine tag, allowing
purification of the recombinant product by a single step of
chromatography on Ni-Sepharose (QiaGen, Chatsworth, CA).
14
CA 02219933 1997-10-31
W O 96/41002 PCTrUS96/08806
Other methods involve the expression o~ recombinant
proteins carrying glutathione-S-transferase se~uences as
tags, allowing purification of the recombinant products on
glutathione affinity columns (Pharmacia Biotech, Uppsala,
Sweden). If necessary, proteins cont~;n;ng purification
tags are then treated so as to remove the tag sequences.
Alternatively, the protein may be isolated from its cell of
origin using st~n~rd protein purification techniques well-
known in the art, including, e.g., molecular sieve, ion-
exchange, and hydrophobic chromatography; and isoelectric
focusing. "Isolation" as used herein denotes purification
of the protein to the extent that it can carry out its
function in the context of the present invention without
interference from extraneous proteins or other contAm;n~nts
derived from the source cells.
The mismatch recognition and modification
proteins used in practicing the present invention may be
derived from any species, from E. coli to hllmAn~, or
mixtures thereof. Typically, functional homologues for a
given protein exist across phylogeny. A "functional
homologue" of a given protein as used herein is another
protein that can ~unctionally substitute ~or the ~irst
protein, either in vivo or in a cell-free reaction.
Mismatch repair proteins:
A number o~ different enzyme systems exist across
phylogeny to repair mismatches that form during DNA
replication. In E. coli, one system involves the MutY gene
product, which recognizes A/G mismatches and cleaves the A-
cont~;n;ng strand (Tsai-Wu et al., ~. Bacteriol.
178:1902,1991). Another system in E. coli utilizes the
coordinated action of the MutS, MutL, and MutH proteins to
recognize errors in newly-synthesized DNA strands
specifically by virtue of their transient state of
n~rm~thylation (prior to their being acted upon by dam
CA 02219933 1997-10-31
W O 96/41002 PCT~US96/08806
methylase in the normal course o~ replication). Cleavage
typically occurs at a h~m; methylated GATC site within 1-2
kb of the mismatch, followed by exonucleolytic cleavage of
the strand in either a 3~-5~ or 5~-3' direction from the
nick to the mismatch. In vivo, this is followed by re-
synthesis involving DNA polymerase III holoenzyme and other
factors (Cleaver, Cell, 76:1-4, 1994).
Mismatch repair proteins for use in the present
invention may be derived ~rom E. coli (as described above)
or from any organism cont~;n;ng mismatch repair proteins
with appropriate functional properties. Non-limiting
examples of use~ul proteins include those derived from
,~7m~e71a ty~h;m77~ium (MutS, MutL); Streptococcus
pneumoniae (HexA, HexB); Saccharomyces cerevisiae ("all-
type", MSH2, MLHl, MSH3); Schizosaccharomyces pombe (SWI4);
mouse (repl, rep3); and human (~all-type", hMSH2, hMLHl,
hPMSl, hPMS2, ducl). Pre~erably, the "all-type" mismatch
repair system from human or yeast cells is used (Chang et
al., Nuc. Acids Res. 19:4761, 1991; Yang et al., J. Biol.
Chem. 266:6480,1991). In a pre~erred embodiment,
heteroduplexes ~ormed between patients' DNA and wild-type
DNA as described above are incubated with human "all-type"
mismatch repair activity that is puri~ied essentially as
described in International Patent Application WO/93/20233.
Incubations are performed in, e.g., lOmM Tris-HCl pH
7.6, lOmM ZnCl2, lmM dithiothreitol, lmM EDTA and 2.9%
glycerol at 37~C for 1-3 hours. In another embodiment,
purified MutS, MutL, and MutH are used to cleave mismatch
regions (Su et al., Proc. Natl. Acad. sci~usA 83:5057,1986;
Grulley et al., J. Biol. Chem. 264:1000,1989).
Nucleotide excision repair proteins:
In E. coli, ~our proteins, designated UvrA, UvrB,
UvrC, and UvrD, interact to repair nucleotides that are
16
CA 02219933 1997-10-31
W O 96/41002 PCT~US96/08806
damaged by W light or otherwise chemically modified
(.~Ant-;3r, Science 266:1954, 1994), and also to repair
mismatches (Huang et al., Proc. Natl . Acad . Sci . USA
91:12213, 1994). UvrA, an ATPase, makes an A2B1 complex
with UvrB, binds to the site of the lesion, unwinds and
kinks the DNA, and causes a conformational change in UvrB
that allows it to bind tightly to the lesion site. UvrA
then dissociates from the complex, allowing UvrC to bind.
UvrB catalyzes an ~n~onllcleolytic cleavage at the fifth
phosphodiester bond 3' from the lesioni UvrC then
catalyzes a similar cleavage at the eighth phosphodiester
bond 5' from the lesion. Finally, UvrD (helicase II)
releases the excised oligomer. In vivo, DNA polymerase I
displaces UvrB and fills in the excision gap, and the patch
is ligated.
In one embodiment of the present invention,
heteroduplexes formed between patients' DNA and wild-type
Dl~TA are treated wlth a r..~xture of IJ-vrA, U-vïB, UVIC, with or
without UvrD. As described above, the proteins may be
purified from wild-type E. coli, or from E. coli or other
a~o~riate host cells cont~;n;ng recombinant genes
encoding the proteins, and are formulated in compatible
buffers and concentrations. The final product is a
heteroduplex cont~;n;ng a single-stranded gap covering the
site of the mismatch.
Excision repair proteins for use in the present
invention may be derived from E. coli (as described above)
or from any organism cont~;n;ng appropriate functional
homologues. Non-limiting examples of useful homologues
include those derived from S. cerevisiae (RAD1, 2, 3, 4,
10, 14, and 25) and hlmn~n~ (XPF, XPG, XPD, XPC, XPA, ERCC1,
and XPB) (Sancar, Science 266:1954, 1994). When the human
' 35 homologues are used, the excised patch comprises an
oligonucleotide ext~n~l~ng 5 nucleotides from the 3' end of
the lesion and 24 nucleotides from the 5' end of the
17
CA 02219933 1997-10-31
W O 96/41002 PCTAJS96/08806
lesion. Aboussekhra et al. (Cell 80:859, 1995) disclose a
reconstituted in vitro system ~or nucleotide excision
repair using puri~ied components derived ~rom human cells.
Chemical Mismatch Recognition:
Heteroduplexes formed between patients' DNA and
wild-type DNA according to the present invention may be
chemically modified by treatment with osmium tetroxide (for
mispaired thymidines) and hydroxylamine (for mispaired
cytosines), using procedures that are well known in the art
(see, e.g., Grompe, Nature Genetics 5:111, 1993; and
Saleeba et al., Meth. Enzymol . 217:288, 1993). In one
embodiment, the chemically modified DNA is contacted with
excision repair proteins (as described above). The
hydroxylamine- or osmium-modified bases are recognized as
damaged bases in need of repair, one of the DNA strands is
selectively cleaved, and the product is a gapped
heteroduplex as above.
Resolvases:
Resolvases are enzymes that catalyze the
resolution of branched DNA intermediates that form during
recombination events (including Holliday structures,
cruci~orns, and loops) via recognition of bends, kinks, or
DNA deviations (Youil et al., Proc.Natl.Acad.Sci.USA 92:87,
1995). For example, ~n~lonl7clease VII derived from
bacteriophage T4 (T4E7) recognizes mismatch regions of from
one to about 50 bases and produces double-stranded breaks
within six nucleotides from the 3' border of the mismatch
region. T4E7 may be isolated ~rom, e.g., a reconbinant E.
coli that overexpresses gene 49 o~ T4 phage (Kosak et
al., Eur. J. Biochem . 194:779, 1990). Another suitable
resolvase ~or use in the present invention is 7~n~7Onllclease
I of bacteriophage T7 (T7E1), which can be isolated using a
18
CA 02219933 1997-10-31
WO 96/41002 PCT~US96/08806
polyhistidine purification tag sequence (M~.Ch~l et al.,
Nature Genetics 9 :177, 1995).
In a preferred embodiment, heteroduplexes formed
between patients' DNA and wild-type DNA as described above
are incubated in a 50 ~1 reaction with 100-3000 units of
T4E7 for 1 hour at 37~C.
3. SEQUENCE DETERMINATION
In practicing the present invention, immobilized
DNA from a patient is hybridized to wild-type DNA to form
mismatch regions and then treated with mismatch repair
proteins, excision repair proteins, resolvases, chemical
modification and cleavage reagents, or combinations of such
agents, to introduce single- or double-stranded breaks at
some predetermined location relative to the site of the
mismatch regions.
In one embodiment, the introduction of single-
stranded breaks at predetermined locations on one or both
sides of a mismatch region causes the selective excision of
a single-stranded fragment covering the mismatch region.
The resulting structure is a gapped heteroduplex in which
the gap may be from about 5 to about 2000 bases in length,
depending on the mismatch recognition system used.
To determine the nucleotide sequence of the
excised region (including the mismatch), the heteroduplexes
are incubated with an appropriate DNA polymerase enzyme in
the presence of dideoxynucleotides. Suitable enzymes for
use in this step include without limitation DNA polymerase
I, DNA polymerase III holoenzyme, T4 DNA polymerase, and T7
DNA polymerase. The only requirement is that the enzyme
'35 be capable of accurate DNA synthesis using the gapped
heteroduplex as a substrate. The presence of
dideoxynucleotides, as in a Sanger sequencing reaction,
19
CA 02219933 1997-10-31
W O 96/41002 PC~r~US96/08806
insures that a nested set of premature t~rm;n~tion products
will be produced, and that resolution of these products by,
e.g., gel electrophoresis will display the DNA sequence
across the gap.
In some circumstances, the sequence obt~;n~ using
this method will correspond to the wild-type strand and not
to the patient's DNA in which the mutation is sought. This
result is easily accomodated by a second round of
sequencing, with or without prior amplification of the
relevant DNA region. In this case, the sequence of the
mutation is determined using as a template the patient's
unmodified DNA in conjunction with sequencing primers
derived from the sequence determined in the first round.
In an alternative embodiment of sequence
det~rm;n~tion, the hybrids formed between the wild-type DNA
and the patient's DNA are then dissociated by denaturation,
and the wild-type DNA and any cleavage products of the
target DNA are removed by w~h;ng. The immobilized
r~m~; n; ng target DNA is then ligated to a synthetic single-
stranded oligonucleotide of predetermined sequence,
designated a "ligation oligonucleotide", that serves as a
primer for enzymatic DNA sequencing. The oligonucleotide
may be from about 15 to about 25 nucleotides in length. A
preferred ligation oligonucleotide has the sequence 5'-
CAGTAGTACAACTGACCCTTTTGGGACCGC-3'. Ligation is achieved
using, e.g., RNA ligase (Pharmacia Biotech, Uppsala,
Sweden).
A typical ligation reaction is performed at 37~C
for 15 min in a 20 ~l reaction cont~;n;ng 50mM Tris-HCl, pH
7.5, lOmM MgCl2, 20mM dithiothreitol, lmM ATP, 100 ~g/ml
bovine serum albumin, at least 1 ~g immobilized target DNA,
a 10-fold molar excess of the ligation oligonucleotide, and
0.1-5.0 units/ml T4 RNA ligase. Following the ligation,
unligated oligonucleotides are removed by w~h;ng~
CA 02219933 1997-10-31
W O 96/41002 PCTrUS96/08806
The sequence o~ DNA ;mmeA;~tely adjacent to the
ligated oligonucleotide is then determined by any method
known in the art. In one embodiment, enzymatic sequencing
is performed according to the dideoxy Sanger technique,
using as a sequencing primer a second oligonucleotide of
predetermined sequence that is complementary to the
ligation oligonucleotide (Sanger et al., Proc. Natl. Acad.
Sci . USA 74:5463, 1977). Each microsequencing reaction is
then resolved by techniques well-known in the art,
including without limitation gel electrophoresis, and the
sequence is determined.
In another embodiment, an oligonucleotide
complementary to the ligated oligonucleotide is used to
prime DNA synthesis using DNA polymerase I in the presence
of all four nucleoside triphosphates. The newly
synthesized strand is then analyzed using hybridization to
oligonucleotide arrays as described in Pease et al., Proc.
Natl . Acad. Sci . USA 91: 5022, 1994.
Identification of a sequence alteration according
to the present invention is preferably achieved in a single
round o~ mismatch recognition and cleavage, oligonucleotide
ligation, and DNA sequencing. This occurs when the ligated
oligonucleotide becomes covalently attached to a) the
immobilized truncated target DNA that contains the
alteration b) within 10-500 bp of either boundary of the
mismatch region. If either o~ these conditions is not
fulfilled, further rounds of sequencing may be required to
localize and identify the sequence alteration. It will be
understood by those of ordinary skill in the art that
- sequencing primers for one or more further rounds of
sequencing will be dictated by the sequence obt~;neA in the
~35 first round (either the same or complementary strands).
Without wishing to be bound by theory, it is contemplated
that one or two sequencing rounds will reveal the
CA 02219933 1997-10-31
W O 96/41002 PCT/U',''.99C~
divergence between a known wild-type sequence and that
contained within the DNA of a particular patient (see
below).
High-Throughput Applications
The methods of the present invention are
particularly suitable for high-throughput analysis of DNA,
i.e., the rapid and simultaneous processing of DNA sam.ples
derived from a large number of patients. Furthe~m~re, in
contrast to other methods for de novo mutation detection,
the methods of the present invention are suitable for the
simultaneous analysis of a large number of genetic loci in
a single reaction; this is designated "multiplex" analysis.
Therefore, for any one sample or for a multiplicity of
samples, the present invention allows the analysis of both
intragenic loci (several regions within a single gene) and
intergenic loci (several regions within different genes) in
a single reaction mixture. The manipulations involved in
practicing the methods of the present invention lend
themselves to automation, e.g., using multiwell microtiter
dishes as a solid support or as a receptacle for, e.g.,
beads; robotics to perform sequential incubations and
washes; and, finally, automated sequencing using
commercially available automated DNA sequencers. It is
contemplated that, in a clinical context, 500 patient DNA
samples can be analyzed within 1-2 days in a cost-effective
m~nne7~ .
Positional Cloning
The methods of the present invention are also
suitable for positional cloning of unknown genes that cause
pathological conditions or other detectable phenotypes in
any organism. "Positional cloning" as used herein denotes
a process by which a previously unknown disease-causing
gene is localized and identified. For example,
CA 02219933 1997-10-31
W O 96/41002 PCTrUS96/08806
identification of multiplex families in which several
members exhibit signs of a genetically-based syndrome often
occurs even when the particular gene responsible for the
syndrome has not been identified. Typically, the search
for the unknown gene involves one or more of the following
time- and labor-intensive steps: 1) cytogenetic
localization of the gene to a relatively large segment of a
particular chromosome; 2) assembly of overlapping cosmid or
Pl clones that collectively cover several hundred thousand
nucleotides corresps~;ng to the identified chromosomal
region; 3) sequencing the clones; and 4) transcript mapping
to identify expressed protein-encoding regions of the gene.
The present invention offers an alternative,
cost-effective method for localizing a disease-causing
gene. Briefly, DNA from affected individuals is hybridized
with normal DNA as described above to form mismatch regions
at the site of the mutation. Preferably, large regions of
DNA corresponding to the chromosomal location are amplified
from the patient's genomic DNA prior to inclusion in the
hybridization reaction. The hybrids are then treated by
any of the methods described above so that mismatch regions
are recognized and cleaved, forming gapped heteroduplexes
across the mismatch region. Finally, the sequence in the
vicinity of the mismatch region is determined.
In this embodiment, det~rm;n~tion of even a short
sequence in the vicinity of the mismatch facilitates
definitive identification of the disease-causing gene.
The short sequence that is determined in the first round of
sequencing can be used to design oligonucleotide probes for
use in screening genomic or cDNA libraries.
-
Other methods in which the primary se~uence
~35 information can be used, either alone or in conjunction
with library screening, include identification of tissue
specific expression, reverse transcription-amplification of
CA 02219933 1997-10-31
W O 96/41002 PCT/U',''.~C~
mRNA, and screening o~ an a~ected population ~or
genotype/phenotype association. Thus, without wishing to
be bound by theory, it is contemplated that a previously
unknown gene that causes a disease or other phenotype can
be quickly and ef~iciently identified by these methods.
The following examples are intended to illustrate
the present invention without limitation.
Example 1: Preparation of Target DNA
A) Preparation of Sample DNA from Blood
Whole blood samples collected in high glucose ACD
Vacutainers~ (yellow top) were centrifuged and the buffy
coat collected. The white cells were lysed with two washes
of a 10:1 (v/v) mixture of 14mM NH4Cl and lmM NaHCO3, their
nuclei were resuspended in nuclei-lysis buffer (10mM Tris,
pH 8.0, 0.4M NaCl, 2mM EDTA, 0.5% SDS, 500 ~g/ml proteinase
K) and incubated overnight at 37~C. Samples were then
extracted with a one-fourth volume of saturated NaCl and
the DNA was precipitated in ethanol. The DNA was then
washed with 70% ethanol, dried, and dissolved in TE buffer
(10mM Tris-HCl, pH 7.5, lmM EDTA).
B) Preparation of Sample DNA from Buccal Cells
Buccal cells were collected on a sterile cytology
brush (Scientific Products) or female dacron swab (Medical
Packaging Corp.) by twirling the brush or swab on the inner
cheek for 30 seconds. DNA was prepared as follows,
;mme~;ately or after storage at room temperature or at 4~C.
The brush or swab was im-m-ersed in 600 ~l of 50mM NaOH
contained in a polypropylene microcentrifuge tube and
vortexed. The tube, still cont~;n;ng the brush or swab,
was heated at 95~C for 5 min, after which the brush or swab
was care~ully removed. The solution cont~;n;ng DNA was
24
CA 02219933 1997-10-31
W O 96/41002 PCT~US96/08806
then neutralized with 60 ~1 o~ lM Tris, pH 8.0, and
vortexed again (Mayall et al., J.Med.Genet. 27:658, 1990).
The DNA was stored at 4~C.
C) A-m~plification o~ Target DNA Prior to Hybridization
DNA from patients with CF was amplified by PCR in
a Perkin-Elmer Cetus 9600 Thermocycler. Five primer sets
were used to simultaneously amplify relevant regions of
exons 4, 10, 20, and 21 of the cystic fibrosis
tr~n~m~mhrane conductance regulator (CFTR) gene (Richards
et al., Hum. Mol.Gen. 2:159, 1993). The 50 ~1 PCR reaction
mix contained the following components: 0.2-l ~g CF
patient DNA, lOmM Tris pH 8.3, 50mM KCl, 1.5mM MgCl2, O.01
(w/v) gelatin, 200~M of each deoxynucleotide triphosphate,
0.4~M of each amplification primer, and 2.5 units of Ta~
polymerase. An initial denaturation was performed by
incubation at 94~C for 20 seconds, followed by 28 cycles of
amplification, each consisting of 10 seconds at 94~C, 10
seconds at 55~C, 10 seconds at 74~C, and a final soak at
74~C ~or 5 min. Following amplification, 8 ~l o~ the PCR
products were electrophoresed in a 2% agarose gel to verify
the presence of all five products.
D) Binding of DNA to a Solid Matrix:
For binding of ampli~ied DNA to a solid support,
the amplification reactions described above are per~ormed
in the present o~ biotinylated primers. The biotinylated
products are then incubated with ~ynabeads~M-280
Streptavidin (Dynal) in a solution cont~;n;ng 10 mM Tris
HCl, pH 7.5, 1 mM EDTA, 2M NaCl, and 0.1% Tween-20 for 15-
30 minutes at 48~C.
CA 02219933 1997-10-31
W O 96/41002 , PCTGUS96/08806
Example 2: Hybridization o~ target DNA and wild-type DNA
A) Preparation of wild-type DNA:
DNA is prepared from blood or buccal cells of
healthy individuals as described in Example 1. A
representative "wild-type~' DNA sample is prepared by
cnmh;n;ng and thoroughly mixing DNA samples derived from
10-200 individuals.
B) Hybridization Reaction:
Hybridizations are carried out in microtiter
dishes contA;n;ng bead-immobilized DNA prepared as in
Example lD above. The hybridization solution contains
approximately 500 ~Lg/ml wild-type DNA (prepared as in
Example 2A above) and approximately 50 ~Lg/ml amplified
immobilized target DNA (prepared as in Example 1) in 10
Tris HCl pH 7.5 - 650I[M NaCl. The reaction mixtures are
heated at 90~C for 3 minutes, ai~ter which hybridizations
are allowed to proceed for 1 hour at 65~C. The
hybridization solution is then removed and the beads are
washed three times in O.lx SSC at 65~C.
C) Blocking of :Eree ends:
The beads cont~;n;ng DNA:DNA hybrids prepared as
described above are treated so that free ends become
blocked and no longer accessible to modification by, e.g.,
RNA ligase. The wells are incubated in 100 ,~Ll of a solution
cont~;n;ng 0.4M potassium cacodylate, 50 mM Tris HCl, pH
6.9, 4 mM dithiothreitol, 1 mM CoC12, 2mM ddGTP, 500 ,ug/ml
bovine serum albumin, and 2 units of terminal transferase
for 15 minutes at 37~C.
26
CA 02219933 1997-10-31
WO 96/41002 PCT~US96/08806
Example 3: Mismatch recognition, cleavage, and sequencing
A) In one embodiment of the present invention,
four identical reactions mixtures, each cont~; n; ng 50 ~l
beads to which DNA hybrids prepared as described in Example
2 are bound, are incubated with 2 ~l of a lOX T4 Polymerase
buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.9, 10 mM MgCl2,
lmM dithiothreitol, and 1 mg/ml bovine serum albumin); 16
~1 water; 1 ~l T4 endonuclease 7 (250-3000 units, obtained
as described in Kosak et al., Eur. J. Biochem. 194:779,
1990); and 1 ~1 T7 DNA polymerase (3 units). The reaction
is allowed to proceed for 1-10 minutes at 37~C.
9 ~l of a ~'termination mix" is then added to each
reaction. '~T~rm;n~tion mix" contains 8 ~M of a single
ddNTP (i.e., ddGTP, ddATP, ddTTP, or ddCTP) and 80 ~M of
all four dNTPs, one of which is labelled with a radioactive
or fluorescent label. In addition, 1 ~l of lOX T4
- polymerase buffer is added, and the reaction is allowed to
proceed for 5 minutes at 37~C.
The reaction mix is removed and the beads are
washed three times with 100 ~1 TE (10 mM Tris-HCl, pH 7.5,
1 mM EDTA). Finally, the beads are resuspended in 6 ~1 gel
loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol
blue, 0.05% Xylene Cyanol FF). The buffer is removed from
the beads and loaded on a 6% denaturing polyacrylamide DNA
sequencing gel.
B) Alternatively, 50 ~l beads cont~;n;ng DNA hybrids
prepared as described in Example 2 are incubated with 500
units of T4 endonuclease 7 in a solution cont~;n;ng 50 mM
Tris-HCl, pH 8.0, 10 mM MgCl2, and 1 mM dithiothreitol for
30 minutes at 37~C. T4 endonuclease 7 is obtained as
described in Kosak et al., Eur. ~. Biochem. 194:779,1990.
CA 02219933 1997-10-31
W O 96/41002 PCT~US96/08806
A~ter the incubation, the beads are heated to
90~C ~or three minutes, after which the solution is quickly
removed and replaced with prewarmed TE, and the beads are
washed three times with TE at room temperature. This
procedure ef~ectively denatures DNA:DNA hybrids and removes
wild-type DNA strands.
Example 4: Mismatch recognition and cleavage using
chemical mismatch cleavage
In one embodiment of the present invention,
microtiter wells prepared as described in Examples 1 and 2
above are treated sequentially with hydroxylamine and
osmium tetroxide.
A) Hyroxylamine treatment:
Hydroxylamine (obtained from Aldrich, Milwaukee,
WI) is dissolved in distilled water, and the pH is adjusted
to 6.0 with diethylamine (Aldrich) so that the final
concentration is about 2.5 M. 200 ~1 of the solution are
incubated within the wells at 37~C ~or 2 hours. The
reaction is stopped by replacing the hydroxylamine solution
with an ice-cold solution cont~;n;ng 0.3 M sodium acetate,
0.1mM EDTA, pH5.2, and 25 ~g/ml yeast tRNA (Sigma, St.
Louis, MO). The wells are then washed in an ice-cold
solution of 10mM Tris-HCl, pH 7.7, lmM EDTA prior to osmium
tetroxide treatment.
B) Osmium tetroxide treatment:
Osmium tetroxide (Aldrich) is dissolved in 10mM
Tris-HCl, pH 7.7, lmM EDTA, and 1.5% (v/v) pyridine to a
concentration o~4% (w/v). The wells are incubated with
this solution ~or 2 hours at 37~C. The reaction is
stopped by replacing the osmium tetroxide solution with an
CA 02219933 1997-10-31
W O 96/41002 PCTAUS96/08806
ice-cold solution cont~;n;ng 0.3 M sodium acetate, 0.lmM
EDTA, pH5.2, and 25 ~g/ml yeast tRNA.
C) Piperidine cleavage:
Chemical cleavage of the C and T bases that react
with hydroxylamine or osmium tetroxide is achieved by
incubating the dishes with lM piperidine at 90~C for 30
min. The wells are then washed extensively with distilled
water.
Example 5: Sequencing of mismatch regions
Immobilized DNAs prepared as described in
Examples 1 and 2 above and subjected to mismatch
recognition and cleavage (as described in Examples 3B or 4
above or by other methods) are incubated with a single-
stranded oligonucleotide having the sequence 5'-
CAGTAGTACAACTGACCCTTTTGGGACCGC-3' under conditions in which
efficient ligation of the oligonucleotide to free 5' ends
is achieved. The oligonucleotide and immobilized DNA are
combined in a solution cont~;n;ng 50 mM Tris HCl, pH 7.5,
10 mM MgCl2, 20 mM dithiothreitol, 1 mM ATP, and 100 ~g/ml
bovine serum albumin, after which RNA ligase (ph~rm~cia
Biotech, Uppsala, Sweden) is added to the solution to
achieve a final enzyme concentration of 0.1-5.0 U/ml.
The reaction is allowed to proceed at 37~C for 15 min.
Following the ligation reaction, the solution is removed,
and the wells are washed with distilled water.
DNA sequencing is then performed using the Sanger
method (Sanger et al.,Proc.Natl.Acad.Sci.USA 74:5463,
1977).
29
CA 02219933 1997-10-31
W O 96/41002 PCTrUS96/08806
Example 6: Positional cloning o~ a disease-causing gene
The experiments described below are performed to
rapidly localize and sequence a genomic region
correspon~;ng to a disease-causing gene.
A multiplex family in which a genetic disease is
expressed is identified using st~n~d clinical indicators.
DNA samples are obtained from affected and unaffected
individuals as described in Example 1 abovei if by patterns
of tr~n~m~sion the disease appears to be an autosomal
recessive syndrome, DNA samples are obtained ~rom those
individuals presumptively heterozygous for the disease
gene.
In one embodiment, all DNA samples are subjected
to mismatch analysis by hybridization to wild-type DNA as
described in Example 2 above. The hybrids are then treated
with mismatch repair proteins to form a gapped
heteroduplex, and the sequence across the gap is determined
as described in Example 3A above.
In an alternative embodiment, all DNA samples are
subjected to mismatch analysis by hybridization to wild-
type DNA as described in Example 2 above. The hybrids are
then treated with T4 ~n~o~llclease 7 as described in Example
3B above. Finally, an oligonucleotide having the sequence
5'-CAGTAGTACAACTGACC~ ~GGACCGC-3~ is ligated to the
cleaved hybrids using RNA ligase, and the products are
subjected to enzymatic DNA sequencing as described in
Example 5 above.
The sequences obtained from unaf~ected, affected,
and presumptively heterozygous family members are compared
with each other and with available sequence databases,
using, ~or example, Sequencher (Gene Codes, Ann Arbor, MI)
and Assembly Lign (Kodak, New Haven, CT) The sequences are
CA 02219933 1997-10-31
WO 96/41002 PCTnJS96108806
also serve as the basis for design of oligonucleotide
probes, which are chemically synthesized and used to probe
human genomic DNA libraries.
,.
