Note: Descriptions are shown in the official language in which they were submitted.
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WO97/10366 PCT~S96/14842
HIGH THROU~l SCREENING METHOD
FOR SEQUENCES OR GENETIC ALTERATIONS IN NUCLEIC ACIDS
Field of the Invention
This invention pertains to high throughput
screening of nucleic acid samples in order to identify the
presence of one or more genetic alterations of interest in
those samples. This invention also pertains to the
identification of specific target nucleic acid sequences
associated with genetic disorders. 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 diagnosis for
genetic counseling. Furthermore, the invention pertains to
specific high-resolution identification of disease-causing
microorganisms.
Background of the Invention
Over the last several years, there has been a
significant increase in the number of identified, cloned,
and characterized genes found to be responsible for
inherited diseases in hllm~n~. As the number of disease-
associated sequences have increased, so has the number ofmutations identified within the genes. In some genes, only
one or a few mutations are specifically responsible for the
disease phenotype (e.g. sickle cell anemia ) (l). However,
in most disease genes, many different causative mutations
exist with no one mutation present at a significant
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frequency within an affected patient population.
Therefore, for both research and clinical diagnostic
applications, improved methods of mutation analysis are
required not only to confirm that a candidate gene truly
represents the disease gene, but also to build mutation
databases and provide clinical diagnostic assays. In
addition, with the increasing number of cancer genes being
discovered (2-4), efficient, cost effective, and highly
informative mutation analysis procedures are necessary in
order to better understand predisposition and polygenic
dlseases .
This has led to the development of two broad
categories of mutation detection technologies (5,6). The
first group, designed to scan for mutations within a gene,
includes single-strand conformational polymorphism (SSCP)
(7), denaturing gradient-gel electrophoresis (D&GE) (8),
heteroduplex analysis (HET) (9), chemical cleavage analysis
(CCM) (10), ribonuclease cleavage (RNAase) (ll), and direct
sequencing of the target (12). Although these procedures
are highly informative, they can be tedious and are
incompatible with high throughput and low cost. Given the
need in the clinical diagnostic laboratory to be able to
analyze large numbers of samples (>500 samples/analysis)
cost effectively, these scanning procedures are not
currently used as routine methods of mutation detection
(5).
In the second group, more direct methods of
mutation analysis have been developed such as allele-
specific amplification (ASA) (13 ), oligonucleotide ligation
assay (OLA) (14), primer extension (15), artificial
introduction of restriction sites (AIRS) (16), allele-
specific oligonucleotide hybridization (ASO) (17), and
variations of these procedures. Together with robotics,
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these methods for direct mutation analysis have helped in
reducing cost and increasing throughput when only a limited
number of mutations need to be analyzed for efficient
diagnostic purposes. However, given that many of the
mutations identified in disease genes are rare, for most
populations undergoing testing, large numbers of mutations
must be analyzed in order to achieve significant detection
frequencies.
Many methods have been developed for the
detection of known mutations. In general, these diagnostic
technologies have been designed to be simple and cost
effective. However, a significant limitation in almost all
of the currently available techniques is the inability to
analyze a large number of samples simultaneously for a
large number of mutations. The most applicable format for
the analysis of large numbers of samples is the dot blot,
wherein the PCR products are bound to a filter membrane and
hybridized with allele-specific probes. However, in a
st~n~rd forward dot blot procedure a separate
hybridization is performed for each allele or mutation of
interest. Therefore, if the number of probes is large,
this procedure becomes cumbersome.
Unfortunately comprehensive multiplex mutation
analysis, for example, >lO0 mutations, cannot readily be
performed by any currently available diagnostic method
while ret~ining the sample throughput and cost
effectiveness needed in a clinical diagnostic laboratory.
Therefore, a significant effort is needed in order to
develop a procedure that will allow large numbers of
samples to be analyzed in a single assay.
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Summary of the Invention
The present invention encompasses high-throughput
methods for detecting and identifying sequences or genetic
alterations (defined as nucleotide additions, deletions, or
substitutions) in a large number of nucleic acid samples,
which is achieved by: immobilizing a plurality of the
nucleic acid samples on a supporti providing a multiplicity
of purine and pyrimidine cont~;n;ng polymers; hybridizing
the immobilized samples with the multiplicity of purine and
pyrimidine contA;ning polymers at substantially the same
time; identifying the hybridized purine and pyrimidine
cont~in;ng polymers wherein the identification of the
hybridized purine and pyrimidine containing polymers
identifies the nucleic acid sequence or one or more genetic
alterations. The hybridized purine and pyrimidine polymers
can be identified by any method well-known in the art, such
as, for example, sequencing, direct labeling, indirect
labeling, and labeling with a unique length marker.
The present invention also encompasses certain
embodiments wherein the sample is not immobilized, and is
reacted with the purine and pyrimidine contAining polymers
in solution (i.e., rather than on a support).
Both the target nucleic acid sequence and/or the
hybridized purine and pyrimidine contAining polymers may be
amplified to facilitate detection and identification. Non-
limiting examples of amplification methods includepolymerase chain reaction (PCR), ligase chain reaction
(LCR), gap-LCR, ligation amplification reaction (LAR),
oligonucleotide ligation assay (OLA), amplification
refractory mutation system (ARMS), competitive
oligonucleotide priming (COP), allele specific PCR, Q-beta
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replicase amplification, nucleic acid sequence based
amplification (NASBA) and branched chain amplification.
Hybridizations can be carried out under conditions that
minimize the differences in melting temperature of hybrids
formed between different purine and pyrimidine contA;ning
polymers and the target nucleic acid sequence.
Brief Description of the Drawings
Figure 1 shows autoradiographic results obtained
from hybridizing multiple identical filters cont~;n;ng
human genomic DNA with 32P-labelled ASOs specific for
different alleles of the cystic fibrosis tr~n~m~mbrane
regulator (CFTR) gene. The ASOs used in each hybridization
are identified on the left of each filter. Lane 1 in each
case contains DNA carrying the mutant sequence
complementary to each ASO; lanes 2-6 contain wild-type
"normal" sequences.
Figure 2A-2D show autoradiographic results
obtained from hybridizing four identical filters cont~;n;~g
human genomic DNA with 32P-labelled ASOs specific for
different alleles of the cystic fibrosis tr~n~m~mhrane
regulator (CFTR) gene. The ASOs used in each hybridization
are identified on the left of each filter. The lanes
marked A contain positive control DNA samples. Rows B-E
contain patient samples analyzed in duplicate, with the
exception of 8C (amplification failure on duplicate
sample), and D7, D8 and E7 (positive controls).
Figures 3A and 3B show the identification of
specific mutations in pool-positive samples identified in
Figure 1. The top row of each filter contains positive
control samples for ASOs in pool 1 and pool 2 as indicated.
Pool 1, lane 1, G542Xi lane 2, G551Di lane 3, R553X; lane
s
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4, W1282X; lane 5, N1303K. Pool 2, lane 1, ~507; lane 2,
R117H; lane 3, 621+1 G->Ti lane 4, S549Ni lane 5, R560T;
lane 6, 1717-1 G->A. Row B contains pool-l or pool-2
positive patient samples. Pool 1, lanes 1 and 2 contain
sample 4, lanes D and E from Figure 1. Pool 2, lanes 1 and
2 contains sample 3, lanes D and E from Figure 2. Lanes 3
and 4 contain sample 5, lanes D and E from Figure 2.
Figure 4 shows a schematic representation of the
methods of the present invention.
Figure 5 depicts general schemes for ligation
based techniques for hybridizing purine and pyrimidine
polymers to immobilized samples and identifying the
hybridized products.
Figure 6 depicts a scheme for identifying
ligation products through chemical cleavage sequencing of
the products, i.e., Maxam-Gilbert sequencing.
Figure 7 depicts a scheme for identifying
ligation products using a conventional Sanger type
sequencing reaction.
Figure 8 depicts another method for identifying
ligation products by Sanger sequencing.
Figure 9 depicts Sanger sequencing of ligation
products that are amplified using a universal priming
sequence.
Figure 10 shows disease gene loci amplified by
multiplex PCR for MASDA assay. DNA samples ~2 mg) were
amplified in multiplexes specific for one of seven
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different genes and analyzed by gel electrophoresis. M=
~X174/HaeIII Molecular weight marker: 1 = Eight amplicon
multiplex within the CFTR gene. 2= Nine amplicon multiplex
within the CFTR gene. 3 = Single amplicon within the ~-
S globin gene. 4 = Two amplicon multiplex within the HEXAgene. 5 = (3+1) multiplex (three amplicon multiplex + one
independent amplicon) for the GCR gene. 6 = Three amplicon
multiplex within the ASPA gene. 7 = Four amplicon multiplex
within the BRCA1 gene. 8 = Three amplicon multiplex within
the FACC gene.
Figure 11 shows simultaneous detection of 106
different mutations within 33 target regions from seven
different genes in a single hybridization assay. Rows 1-7
= Detection of 66 CFTR mutations present in cystic fibrosis
(CF). Rows 8-9 = Negative control samples for cystic
fibrosis (CF-) mutations. Rows 10-11 = Detection of 14 ~-
globin mutations in ~-thalassemia (~Thal.) and 2 ~-globin
mutations in sickle cell anemia (SCA). Row 12 = Detection
of 3 HEXA mutations present in Tay Sachs (TSD). Row 13 =
Detection of 8 GCR mutations present in Gaucher's disease
(GCR). Row 14 = Detection of 4 ASPA mutations present in
Canavan Disease (CD). Row 15 = Detection of 5 BRCA 1
mutations present in breast cancer (BRC). Row 16 =
Detection of 4 FACC mutations present in Fanconi anemia
~FA). Disease-specific negative control samples follow the
positive samples in Rows 11-16.
Figure 12 shows the band patterns generated by
chemical cleavage of eluted ASOs and reveals the identity
of the mutation. C = C cleavage reaction; G = G cleavage
reaction.
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Figure 13 shows the band patterns generated by an
enzymatic sequencing procedure reveal the identity of the
mutation. The ASO(s) eluted from mutation-positive samples
were used to prime cycle sequencing reactions in a complex
mixture of templates. Each template contained a region
complementary to a specific ASO (the priming site), a
common "stuffer region" (Region B) and a downstream
mutation-specific identifier sequence (Region A). With
limited C and G sequencing (Lanes C and G for each sample),
the fingerprint generated from the mutation-specific
identifier sequence unequivocally identified the specific
ASO, and therefore the mutation present in the target DNA.
CF = cystic fibrosis; BT = ~-thalassemia; BRC = Breast
Cancer Susceptibility BRCA1 gene. Lane 1 = CF17 mutation;
Lane 2 = CF 20 mutation; Lane 3 = BT5 mutation; Lane 5 = CF
negative control; Lane 6 = BT negative control; Lane 7 =
BRC negative control.
Figure 14 shows the simultaneous detection of 106
different mutations in over 500 different patient samples
in a single ASO hybridization assay.
Detailed Description of the Invention
All patent applications, patents, and literature
references cited in this specification are hereby
incorporated by reference in their entirety. In case of
conflict, the present description, including definitions,
will control.
Definitions:
1. An "allele-specific oligonucleotide" (ASO) as
defined herein is an oligonucleotide having a sequence that
is identical or almost identical to a known nucleic acid
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W097tlO366 PCT~S96/14842
portion. Often, an ASO contains a small change relative to
the prevalent "wild type" sequence. This change may
comprise addition, deletion, or substitution of one or more
nucleotides. ASOs can be designed to identify any
addition, deletion, or substitution, as long as the nucleic
acid sequence is known.
2. A "variant" sequence as used herein
encompasses a nucleic acid sequence that differs from a
known sequence by the addition, deletion, or substitution
of one or more nucleotides.
3. "Amplification" of a nucleic acid sequence as
used herein denotes the use of polymerase chain reaction
(PCR) (Saiki et al., Science 239:487, 1988), ligase chain
reaction (Barany, Proc. Natl. Acad. Sci. USA 88:189, 1991)
(LCR), gap-LCR (Abravaya et al., Nuc. Acids Res. 23:675,
1995), ligation amplification reaction (Wu et al., Genomics
4:560, 1989); ASPCR (Wu et al., Proc. Natl. Acad. Sci. USA
86:2757, 1989), ARMS (Newton et al., Nucl. Acids Res.
17:2503, 1989), or other methods to increase the
concentration of a particular nucleic acid sequence.
4. "Chemical sequencing" of nucleic acids
denotes methods such as that of Maxim and Gilbert (Maxim-
Gilbert sequencing, Maxam and Gilbert, 1977, Proc. Natl.
Acad. Sci. USA 74:560), in which nucleic acids are r~n~omly
cleaved using individual base-specific reactions.
5. ~Enzymatic sequencing" of nucleic acid
denotes methods such as that of Sanger (Sanger et al.,
1977, Proc. Natl. Acad. Sci. USA, 74:5463), in which a
single-stranded DNA is copied and r~n~omly t~rm'n~ted using
DNA polymerase.
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6. In this specification, the terms "bound" and
"hybridized~ are used interchangeably to denote the
formation of nucleic acid:purine and pyrimidine cont~in;ng
polymer duplexes. The term "affinity purified" denotes
S purification using hybridization.
7. "High-throughput" denotes the ability to
simultaneously process and screen a large number of nucleic
acid samples (e.g., in excess of 50 or lO0 nucleic acid
samples) and a large number of target sequences within
those samples.
8. "Purine and pyrimidine containing polymers~ is
meant to include DNA, RNA and other polymers cont~ining
purines and pyrimidines that are capable of Watson-Crick
base pairing, and which do not necessarily carry a sugar
phosphate backbone, such as PNA. See, J. Am. Chem. Soc.,
114:1895-97 (1992).
The present invention provides refin~mPnts of a
modified allele-specific oligonucleotide approach for the
simultaneous analysis of large numbers of patient samples
for multiple CF mutations (25). Invention methods further
provide a Multiplex Allele-Specific Diagnostic Assay
(MASDA), which has the capacity to cost effectively analyze
large numbers of samples (>500) for a large number of
mutations (>lO0) in a single assay. Like the more familiar
'chip' technologies (19-22), MASDA uses oligonucleotide
hybridization to interrogate DNA sequences. However, in
contrast to many oligonucleotide array approaches, in the
invention MASDA technology, the target DNA is immobilized
to the solid support, and interrogated in a combinatorial
fashion with a pool of ASOs (i.e. a single mixture of
mutation-specific oligonucleotides) in solution. By
ret~i n; ng the forward dot blot format, it is possible to
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simultaneously analyze large numbers of samples (>500) for
a large number of mutations (>l00). In phase I of the
combinatorial analysis, the ASO(s) corresponding to the
specific mutation(s) present in a given sample is hybrid-
selected from the pool by the target DNA. Followingremoval of unhybridized ASOs, sequence-specific band
patterns associated with the bound ASOs are generated by
chemical or enzymatic sequencing, and the mutation or
mutations present in the sample are easily identified.
Using the gene targets CFTR (26), $-globin (l), HEXA (27),
GCR (28), ASPA (29), BRCAl (3), and FACC (30) as a model
system, we demonstrate that MASDA not only allows different
patient samples with different disease indications to be
analyzed in a single assay, but allows the identification
of multiple mutations within a single gene or multiple
genes in a single patient's DNA sample.
The present invention encompasses a high-
throughput method for screening nucleic acid samples for
target sequences or sequence alterations and, more
particularly, for specific DNA sequences in DNA isolated
from a patient. The method is applicable when one or more
genes or genetic loci are targets of interest. It will
also be appreciated that this method allows for rapid and
economical screening of a large number of nucleic acid
samples for target sequences of interest.
In one embodiment, the specific nucleic acid
sequence comprises a portion of a nucleic acid, a
particular gene, or a genetic locus in a genomic DNA known
to be involved in a pathological condition or syndrome.
Non-limiting examples include cystic fibrosis, sickle-cell
anemia,~-thalassemia, and Gaucher's disease.
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In another embodiment, the specific nucleic acid
sequence comprises part of a particular gene or genetic
locus that may not be known to be linked to a particular
disease, but in which polymorphism is known or suspected.
In yet another embodiment, the specific nucleic
acid 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.
In accordance with the present invention, the
target nucleic acid represents a sample of nucleic acid
isolated from a patient. This nucleic acid 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, fetal cells, or any cells present in
tissue obtAlne~ by biopsy. Body fluids can include blood,
urine, cerebrospinal fluid, semen, and tissue exudates at
the site of infection or inflammation. Nucleic acids can be
extracted from the cell source or body fluid using any of
the numerous methods that are standard in the art. It will
be understood that the particular method used to extract
the nucleic acid will depend on the nature of the source.
The minimum amount of DNA, for example, that can be
extracted for use in a preferred form of the present
invention is about 5 pg (corresponding to about l cell
equivalent of a genome size of 4 x l09 base pairs).
Once extracted, the target nucleic acid may be
employed in the present invention without further
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manipulation. Alternatively, one or more specific regions
present in the target nucleic acid may be amplified by PCR.
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 nucleic acid
sequences within the target sequence population. The length
of nucleic acid sequence that can be amplified ranges from
80 bp to up to 30 kbp (Saiki et al., 1988, Science,
239:487).
In one embodiment, the target nucleic acid, with
or without prior amplification of particular sequences, is
bound to a solid phase or semi-solid phase matrix. This
allows for the simultaneous processing and screening of a
large number of nucleic acid samples from different
sources. 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, polymer gels, agarose and the like. It
will be understood by a skilled practitioner that the
method by which the target nucleic acid is bound to the
matrix will depend on the particular matrix used. For
example, binding to nitrocellulose can be achieved by
simple adsorption of nucleic acid to the filter, followed
by baking the filter at 75-80C under vacuum for 15 min-2h.
Alternatively, charged nylon membranes can be used that do
not require any further treatment of the bound nucleic
acid. Beads and microtiter plates that are coated with
avidin can be used to bind target nucleic acid that has had
biotin attached (via e.g. the use of biotin-conjugated PCR
primers). In addition, antibodies can be used to attach
target nucleic acid to any of the above solid supports by
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coating the surfaces with the antibodies and incorporating
an antibody-specific hapten into the target nucleic acid.
While immobilization of the target nucleic acid
is generally preferred, in some embodiments it may be
desirable to hybridize the polymers to the target in
solution, i.e., without having bound the target to a
support. For example, polymers may be hybridized to the
target in solution, and then ligated in solution as part of
a technique according to the invention for high throughput
screening. Ligation techniques are discussed further
below.
In practicing the present invention, the
untreated or amplified target nucleic acid, preferably
bound to a solid phase or semi-solid phase matrix, is
incubated with a mixture of purine and pyrimidine
cont~ining polymers (hereinafter also referred to as
"polymer~ or "polymers"). These polymers are preferably
allele-specific oligonucleotides (ASOs). 10-200 ASOs can
be pooled for a single hybridization, preferably 50-100,
and most preferably 50. The length of individual ASOs may
be 16-25 nucleotides, preferably 17 nucleotides in length.
The purine and pyrimidine cont~inin~ polymers may
be synthesized chemically by methods that are st~n~rd in
the art, e.g., using commercially available automated
synthesizers. These polymers may then be radioactively
labelled (e.g. end-labelled with 32p using polynucleotide
kinase) or conjugated to other commonly used "tags" or
reporter molecules. For example, fluorochromes (such as
FITC or rhodamine), enzymes (such as alkaline phosphatase),
biotin, or other well-known labelling compounds may be
attached directly or indirectly. Furthermore, using
st~n~rd methods, a large number of r~n~omly permuted
14
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WO97/10366 PCT~S96/14842
polymers can be synthesized in a single reaction. As
detailed below, the present invention does not require that
individual hybridizing sequences be determined prior to the
-hybridization. Rather, the sequence of bound polymers can
be determined in a later step.
As described in co-pending U.S. patent
application Serial No. 07/957,205 (filed October 6, 1992)
and in Shuber et al., 1993, Human Molecular Genetics,
2:153-158, the hybridization reaction can be performed
under conditions in which polymers such as those cont~; n i ng
different sequences hybridize to their complementary DNA
with equivalent strength. This is achieved by: 1)
employing polymers of equivalent length; and 2) including
in the hybridization mixture appropriate concentrations of
one or more agents that eliminate the disparity in melting
temperatures among polymers of identical length but
different guanosine+cytosine (G+C) compositions. Agents
that may be used for this purpose include without
limitation quaternary ammonium compounds such as
tetramethylammonium chloride (TMAC).
TMAC acts through a non-specific salt effect to
reducing hydrogen-bonding energies between G-C base pairs.
At the same time, it binds specifically to A-T pairs and
increases the thermal stability of these bonds. These
opposing influences have the effect of reducing the
difference in bonding energy between the triple-hydrogen
bonded G-C based pair and the double-bonded A-T pair. One
consequence, as noted above, is that the melting
temperature of nucleic acid to nucleic acid hybrids formed
in the presence of TMAC is solely a function of the length
of the hybrid. A second consequence is an increase in the
slope of the melting curve for each probe. Together these
- 35 effects allow the stringency of hybridization to be
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increased to the point that single-base differences can be
resolved, and non-specific hybridization min;m;zed ~Wood et
al., 1985, Proc. Natl. Acad. Sci., USA 82:1585.).
S It will be apparent to those skilled in the art
that any agent that exhibits these properties can be used,
if desired, in practicing the present invention. Such
agents can be easily identified by detPrm;n;ng melting
curves for different test oligonucleotides in the presence
and absence of increasing concentrations of the agent.
This can be achieved by attaching a target nucleic acid to
a solid matrix such as a nylon filter, individually
hybridizing radiolabelled oligonucleotides of identical
length but different G+C compositions to the filter,
washing the filter at increasing temperatures, and
measuring the relative amount of radiolabelled probe bound
to the filter at each temperature. An agent that, when
present in the hybridization and washing steps described
above, results in approximately superimposable and steep
melting curves for the different oligonucleotides may be
used.
In practicing the present invention, the target
nucleic acid and polymers can be incubated for sufficient
time and under appropriate conditions to achieve maximal
specific hybridization and min;m~1 non-specific, i.e.
background, hybridization. The conditions to be considered
include the concentration of each polymer, the temperature
of hybridization, the salt concentration, and the presence
or absence of unrelated nucleic acid. It will further be
appreciated that the polymers can be separated into at
least two groupings, each grouping cont~ining a sufficient
number of polymers to allow for hybridization. For
example, it may be preferred to divide the total number of
polymers of a pool to be hybridized to the nucleic acid
16
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WO97/10366 PCT~S96/14842
samples into groupings of about 50 polymers. Each group of
polymers can be hybridized to the nucleic acid immobilized
on the support in a sequential manner, but the polymers
comprising each group can be hybridized to the nucleic acid
at substantially the same time. Additionally, immobilized
nucleic acid samples may be hybridized to at least one pool
of polymers, the identity of the hybridizing polymers
determined, and then the nucleic acid samples hybridized
again with the same or different polymer pools.
The concentration of each purine and pyrimidine
cont~;n;ng polymer generally ranges from 0.025 to 0.2 pmol
per ml of hybridization solution. When polymers of known
sequence are used, the optimal concentration for each
polymer can be determined by test hybridizations in which
the signal-to-noise ratio (i.e. specific vs. non-specific
b;n~;ng) of each polymer is measured at increasing
concentrations of labeled polymer. To further reduce
background hybridization, oligonucleotides containing the
unlabeled non-variant ~i.e. wild-type) sequence may be
included in the reaction mixture at a concentration
equivalent to l-lO0 times the concentration of the labelled
polymer.
The temperature for hybridization can be
optimized to be as high as possible for the length of the
polymers being used. This can be determined empirically,
using the melting curve determination procedure described
above. It will be understood by skilled practitioners that
det~rm;n~tion of optimal time, temperature, polymer
concentration and salt concentration should be done in
concert.
It is intended that the hybridized polymers
identified by the invention be those that are perfectly
17
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WO97/10366 PCT~S96/14842
hybridized. Methods described above minimize imperfect
hybridization. Such methods, however, are not always
necessary. In the ligation procedure described in the
Examples, imperfect hybrids may form, but perfect hybrids
are selectively identified.
Following hybridization, unbound polymers are, if
necessary, removed such as by washing the matrix-bound
nucleic acid in a solution cont~i n ing TMAC or similar
compounds, under conditions that preserve perfectly matched
nucleic acid:polymer hybrids. Washing conditions such as
temperature, nature and concentration of salts, and time of
washing, are determined empirically as described above. At
this stage, the presence of bound polymers may be
determined. Different methods for detection will depend
upon the label or tag incorporated into the polymers. For
example, radioactively labelled or chemiluminescent ASOs
that have bound to the target nucleic acids can be detected
by exposure of the filter to X-ray film.
Alternatively, polymers cont~ining a fluorescent
label can be detected by excitation with a laser or lamp-
based system at the specific absorption wavelength of the
fluorescent reporter. Still further, polymers can each
carry, in addition to the probe sequence, a molecular
weight modifying entity (MWME) that is unique for each
member of the polymer pool. The MWME does not participate
in the hybridization reaction but allows direct
identification of the separated polymer by det~rmin~tion of
the relative molecular weight by any number of methods.
Other methods for detection and identification are
described below.
In an optional subsequent step, the bound
polymers are separated from the matrix-bound target nucleic
18
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W097/10366 PCT~S96/14842
acid. Separation may be accomplished by any means known in
the art that destabilizes nucleic acid to polymer hybrids,
i.e. lowering salt concentration, raising temperature,
exposure to formamide, alkali, etc. For example, the bound
polymers may be separated by incubating the target nucleic
acid-polymer complexes in water, and heating the reaction
above the melting temperature of the nucleic acid:polymer
hybrids. This obviates the need for further treatment or
purification of the separated polymers.
According to this invention, the hybridized
polymers, with or without separation from the target
nucleic acid, can be identified by a number of different
methods that will be readily appreciated by those of skill
in the art. By sequencing the polymers, it is possible to
correspondingly identify target sequences or genetic
alterations in the nucleic acid samples.
The polymers can also be identified by directly
labeling them with a unique reporter that provides a
detectable signal. Polymers that are directly labeled can
be detected using radioactivity, fluorescence, colorimetry,
x-ray diffraction or absorption, magnetism, enzymatic
activity, chemilt]~inescence, and electrochemiluminescence,
and the like. Suitable labels include fluorophores,
chromophores, radioactive atoms ~such as 32p and 125I),
electron dense reagents, and enzymes that produce
detectable products. See L. Kricka, Nonisotopic DNA Probe
Techniques, Chapters l and 2, Academic Press, 1992
(hereinafter "Kricka").
In addition to direct labeling o~ polymers,
- indirect labeling may also be used. Many binding pairs are
known in the art for indirect labeling, including, for
- 35 example, biotin - avidin, biotin - streptavidin, hapten -
19
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WO97/10366 PCT~S96/14842
antihapten antibody, sugar - lectin, and the like. When
used with the present invention, one member of a binding
pair can be attached to the polymer and the other member of
the binding pair directly labeled as described above.
Subsequent to hybridization, the polymers that are bound to
target nucleic acid sequences may be identified by
incubation with the labeled member and subsequent detection
of the binding pair-label complex. See, Bioconjugate
Chemistry, 1:165-187 (l990)i Kricka, Chapters 1 and 2.
The polymers can still further be identified by
using unique length markers. That is, by providing
polymers having components that contribute a predetermined
and unique molecular weight to each individual polymer, in
addition to the portions that participate in hydrogen
bonding interactions with target nucleic acids, it is
possible to identify an individual polymer by molecular
weight. See, e.g., Nucleic Acids Res., 22:4527-4534
(1994).
Still further, hybridized polymers can be
identified by use of hybridization arrays. In such arrays,
purine and pyrimidine cont~ining polymers of predetermined
sequence are immobilized at discrete locations on a solid
or semi-solid support. When used with the present
invention, the sequence of each immobilized polymer
comprising the array is complementary to the sequence of a
member of the polymer pool. Members of the polymer pool
that hybridize with target nucleic acids can be identified
after separation from target nucleic acids by
rehybridization with immobilized polymers forming the
array. The identity of the polymer is determined by the
location of hybridization on the array. See, U.S. Patent
No. 5,202,231 and WO 8910977.
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WO97/10366 PCT~S96/14842
Other permutations and possibilities will be
readily apparent to those of ordinary skill in the art, and
are considered as equivalents within the scope of the
instant invention.
More particularly, in one embodiment, the
hybridized polymer is directly subjected to sequencing,
using a chemical method st~n~rd in the art (e.g. Maxam-
Gilbert sequencing, Maxam and Gilbert, 1977, Proc. Natl.
Acad. Sci., USA, 74:560). This method does not require
that polymers be separated from the target nucleic acid
prior to sequencing, and, further, is particularly
applicable when randomly permuted mixtures of polymers are
used.
In another embodiment, the hybridized polymers
are identified by enzymatic sequencing (Sanger et al.,
1977, Proc. Natl. Acad. Sci., USA, 74:5463). In this case,
oligonucleotides are synthesized that contain sequences
complementary to the polymers and additional pre-determined
co-linear sequences that act as sequence "tags" (see
Example 4 below). Separation of the polymers from the
target nucleic acid is performed in the presence of a
mixture of these complementary, "tagged" oligonucleotides.
When incubated under Sanger sequencing conditions (see e.g.
Example 5 below), the polymers hybridize to their
complementary sequences and act as primers for the
sequencing reaction. Det~rm;n~tion of the resulting primed
sequence "tag" then identifies the polymer(s) present in
the reaction.
In a further embodiment, the hybridized polymers
are incubated with complementary oligonucleotides that may
contain universal primer sequences and/or a sequencing
primer sequence with or without an additional "tag"
21
CA 0220~234 1997-0~-13
WO97/10366 PCT~S96/14842
sequence (see Example 4 below). In both cases, initial
hybridization of a polymer to its complementary
oligonucleotide allows the polymer to serve as the initial
primer in a single extension reaction. In one case, the
extension product is then used directly as template in a
cycle sequencing reaction. Cycle sequencing of the
extension products results in amplification of the
sequencing products. In designing the complementary
oligonucleotides, the sequencing primer is oriented so that
sequencing proceeds through the polymer itself, or,
alternatively, through the "tag" sequence.
In the second case, the extension product
includes a universal primer sec~uence and a sequencing
primer sequence. This extension product is then added to a
linear amplification reaction in the presence of universal
primer. The oligonucleotides cont~;ning complementary
sequences to bound polymers are therefore selectively
amplified. In a second step, these amplified sequences are
subjected to Sanger sequencing, using the built-in
sequencing primer sequence. In this case, the sequencing
primer is placed immediately upstream of a "tag" sequence
as above. Thus, determination of the "tag" sequence will
identify the colinear polymer sequence.
In practicing the present invention, it is not
necessary to determine the entire sequence of the polymer
or of a complementary tagged oligonucleotide. It is
contemplated that l, 2, or 3 sequencing reactions (instead
of the four needed to obtain a complete sequence) will be
effective in producing characteristic patterns (similar to
"bar codes~) to allow the ;mme~iate identification of
individual polymers. This approach is applicable to m~n~
sequencing methods using radioactively labelled polymers,
which produce analog or digitized autoradiograms, as well
22
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WO97/10366 PCT~S96/14842
as to automated sequencing methods using non-radioactive
reporter molecules, which produce digitized patterns. In
- either case, comparisons to an established data base can be
performed electronically. Thus, by reducing the number of
required sequencing reactions, the methods of the present
invention facilitate the economical analysis of multiple
samples, and of multiple nucleic acid sequences or genetic
alterations within each sample.
The present invention accommodates the
simultaneous screening of a large number of potential
polymers in a single reaction. In practice, the actual
num.ber of polymers that are pooled for simultaneous
hybridization is determined according to the diagnostic
need. For example, in cystic fibrosis (CF), one particular
mutation (A508) accounts for more than 70% of CF cases.
Thus, a prelimin~ry hybridization with a labelled or tagged
~508-specific polymer according to the present methods,
followed by detection of the bound polymer, will identify
and eliminate ~508 alleles. In a second ("phase two")
hybridization, a large number of polymers encoding other,
less frequent, CF alleles are utilized, followed by
separation and sequencing as described above.
In other clinical situations, however, a single
mutation that appears with as high a frequency as the ~508
mutation in CF does not exist. Therefore, pools of
polymers are determined only by the number of independent
hybridizations that would be needed in a phase two analysis
on a pool positive sample.
-
In addition, in current clinical practice,different clinical syndromes, such as cystic fibrosis, ~-
23
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WO97/10366 PCT~S96/14842
thalassemia, and Gaucher's disease, are screened
independently of each other. The present invention, by
contrast, accommodates the simultaneous screening of large
numbers of nucleic acid samples from different sources,
including different m~mm~ls, with a large number of
polymers that are complementary to mutations in more than
one potential disease-causing gene.
In the same manner, when clinical indicators
suggest infection by a foreign agent or microorganism, the
present invention provides for simultaneous screening for a
large number of potential foreign nucleic acids.
Furthermore, particular strains, variants, mutants, and the
like of one or more microorganisms can also be
distinguished by employing appropriate polymers in the
screening.
The methods of the present invention also make it
possible to define potentially novel mutant alleles carried
in the nucleic acid of a patient or an invading
microorganism, by the use of r~n~omly permuted polymers in
phase one or phase two screening. In this embodiment,
separation of the bound polymers, followed by sequencing,
reveals the precise mutant sequence.
This invention further contemplates a kit for
carrying out high-throughput screening of nucleic acid
samples according to this invention. The kit will include,
in packaged combination, at least the following components:
a support, a multiplicity of purine and pyrimidine
cont~i ni ng polymers, appropriate labeling components, and
enzymes and reagents required for polymer sequence
det~rmin~tion.
24
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WO97/10366 PCT~S96/14842
Model Systems of the Invention
-
Seven different gene targets, representing eight
different diseases, were chosen as a model system for
complex mutation detection (Table 1). A total of 106
different mutations were analyzed in a single hybridization
and detection procedure, referred to as MASDA 106.
Although large numbers of mutations have been identified
within the majority of disease genes listed, for the
purposes of this study a selected number of these mutations
were used (Table 1 columns 3 and 4). The specific
mutations chosen within each disease gene represented the
most clinically relevant for diagnostic applications The
largest number of mutations analyzed resided within the
CFTR gene. In addition to the most frequently detected
mutations within a CF patient population, additional point
mutations were included that lead to premature translation
termin~tion, and subsequently a truncated protein product.
A total of 33 different amplification products were needed
in order to interrogate for the presence or absence of the
106 different mutations (Table 1 column 5). It is
important to note that amplifications were performed in a
disease-specific manner only. In other words, if a patient
was suspected to be a cystic fibrosis carrier, the DNA
sample was amplified for the cystic fibrosis gene only.
The specific mutations examined within each
disease gene are shown in Tables 1-8. These tables also
include the size (bp) of regions amplified, and the primers
used for each amplification.
- Disease Specific Target Amplifications
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WO97/10366 PCT~S96/14842
Where applicable, multiplex PCR was performed to
reduce the number of PCR reactions needed (Table l, column
5). A total of 9 reactions facilitated amplification of 33
different loci. All single or multiplex PCR reactions were
performed in a disease-specific manner. In other words,
DNA samples were amplified for the relevant disease gene
only, and not for all loci examined in the assay. Examples
of the various disease-specific amplification products are
shown in Figure lO. In order to include 66 CF mutations
within the study discussed herein, 17 different regions
within the CFTR gene were amplified using two multiplex PCR
reactions (Figure lO lanes l and 2). Amplicon sizes ranged
from 130 - 510 bp. A single amplification product of 1600
bp was sufficient to include 14 ~-thalassemia and two
sickle cell anemia associated mutations within the ~-globin
gene (Figure lO lane 3). For Tay Sachs, a 2-plex
amplification reaction was designed to examine 3 mutations
(Figure lO lane 4). To examine Canavans-associated
mutations 3-plex amplification reactions were performed
(Figure lO lane 6). A separate 4-plex amplification was
performed for five Breast Cancer Susceptibility-related
mutations (Figure lO lane 7) and a single 3-plex
amplification for Fanconi Anemia-associated mutations
(Figure lO lane 8). For Gauchers disease, the GCR
pseudogene neccesitated a 3-plex amplification and an
independent, single amplicon amplification. For convenience
of analysis, aliquots from both reactions were pooled and
electrophoresed in the same lane of the analytical gel
(Figure lO lane 5).
Mutation Detection
In order to simultaneously analyze large numbers
of samples for the mutations listed in Table l, the
st~n~rd forward dot blot format was employed and a single
26
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WO97/10366 PCT~S96/14842
multiplex hybridization was performed (Figure 11).
Although the percent G-C content of the 106 mutation-
specific oligonucleotides ranged between 18% and 76%, the
use of tetramethylammonium chloride (TMAC) (31) allowed all
106 mutation-specific oligonucleotides to be mixed together
and hybridized in a single pool. Furthermore, the presence
of TMAC in the hybridization and wash solutions allowed the
hybridization and washes to be performed at the same
temperature. As seen in Figure 11, only the 106 mutation-
specific positive control samples generated signals uponautoradiography with no significant non-specific signal
exhibited by the negative control samples. Overall signal
intensities and signal-to-noise-ratios generated for the
different mutation-specific positive control samples were
optimized by adjusting concentrations of each mutation-
specific oligonucleotide within the hybridization.
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WO97/10366 PCT~S96/14842
Mutation Identification
The specific mutation present within any pool-
S positive sample was identified by eluting the hybridized
oligonucleotide from the sample DNA and directly
interrogating the oligonucleotide sequence. In one scheme,
the eluted oligonucleotides were attached to a solid
support, G and C base specific chemical modification
reactions were performed, and the reaction products
separated by polyacrylamide gel electrophoresis (32).
Figure 12 represents an example of the CG limited
sequencing fingerprints produced from some of the
oligonucleotides eluted from the pool-positive control
samples in Figure 3 (CF30, CF31, TSl, TS2, TS3, BT2, BT3*,
BT6 and BT7). Each eluted oligonucleotide shown in Figure
12 generated a characteristic fingerprint which
unambiguously identified the specific mutation present in
the pool-positive sample DNA. Unique band patterns were
generated for each of 106 ASOs. Sequence analysis of all
106 eluted oligonucleotides verified that a positive result
from the single pooled hybridization represented specific
oligonucleotide hybridization with no significant cross
hybridization between different probes. In addition, one
sample cont~ining two ~-globin mutations (Figure 12, lane
BT3*) generated a unique fingerprint made up of two
superimposed oligonucleotide-specific band patterns. This
demonstrated that a compound heterozygote genotype was
readily identified using this technique.
In addition to the chemical modification and
cleavage procedure, an enzymatic protocol for eluted
oligonucleotide identification was developed. This
procedure involved using the eluted mutation specific
oligonucleotide as a primer in a cycle sequencing reaction.
28
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WO97/10366 PCT~S96/14842
The eluted oligonucleotide was added to a cycle sequencing
reaction mix cont~inlng a mixture of synthetic (77-mer)
templates. Each synthetic template contained a different
priming sequence complementary to one of the mutation-
specific oligonucleotides present in the pooledhybridization reaction, and a downstream specific
identifier sequence to generate unique, mutation-specific
fingerprints identifying the eluted ASO. Figure 13 is an
example of the C and G band patterns generated from cycle
sequencing reactions utilizing oligonucleotides eluted from
positive (mutant genotype CF17, CF20, BT5 and BRC5) and
negative (normal genotype CF neg., BT neg., and BRC neg.)
control samples. Each reaction performed with
oligonucleotides eluted from positive control samples
(Figure 13 lanes 1-4) generated a common band pattern
contained within all synthetic templates (Region B)
followed by a mutation-specific fingerprint (Region A). The
pattern observed in the mutation-specific fingerprints
allowed unequivocal identification of the corresponding ASO
primer, and consequently the specific mutation present in
the patient sample. No band patterns were observed when
cycle sequencing reactions were performed with eluates from
negative control samples (Figure 13 lanes 5-7).
Large Scale Sample Analysis
To validate the invention MASDA procedure, a
blinded analysis was performed to assess the ability of the
MASDA technique to identify mutations as envisioned in the
diagnostic setting. Figure 14 represents the hybridization
results generated from analyzing >500 different DNA
samples for the presence of 106 different mutations, in a
single hybridization assay. All samples known to carry one
of the 106 different mutations were correctly identified as
- 35 pool-positive in the multiplex hybridization (Figure 14).
29
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W097/10366 PCT~S96/14842
The specific mutations were correctly identified within
each pool-positive sample by performing the chemical
modification and cleavage procedure. It is significant to
note that no increase in non-specific background was
observed when sample throughput was increased to more than
500 samples in a single hybridization assay.
The following examples are intended to further
illustrate the present invention without limiting the
invention thereof.
Example l: Mutation-Positive Genomic DNA and Genomic
Negative DNA Samples
A) Preparation of Sample DNA from Blood
Whole blood samples collected in high glucose ACD
VacutainersTM (yellow top) were centrifuged and the buffy
coat collected (33). The white cells were lysed with two
washes of a lO:l (v/v) mixture of 14mM NH4Cl and lmM
NaHC03, their nuclei were resuspended in nuclei-lysis
buffer (lOmM Tris, pH 8.0, 0.4M NaCl, 2mM EDTA, 0.5% SDS,
500 ~g/ml proteinase K) and incubated overnight at 37C.
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 (lOmM 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, immediately or after storage at room temperature
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WO97/10366 PCT~S96/14842
or at 4C. The brush or swab was immersed 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 95C for 5 min, after which the brush or swab
was carefully removed. The solution cont~'n;ng DNA was
then neutralized with 60 ~l of lM Tris, pH 8.0, and
vortexed again (Mayall et al., J.Med.Genet. 27:658, l990).
The DNA was stored at 4C.
C) Cloned Positive Control DNA Samples
When mutation-positive genomic DNA was not
available, oligonucleotides representing 40 bp of
endogenous gene sequence including the mutation were
synthesized, cloned into pGEM~-3Zf(+) vectors (Promega
Corporation, Madison, WI), and the presence of the mutation
in each clone verified by sequencing.
D) Amplification of Target DNA Prior to Hybridization
DNA Amplifications
As a model system for complex mutation detection,
mutations were selected from 33 regions in seven different
genes. The genes included the cystic fibrosis
tr~n~m~mhrane conductance regulator gene (CFTR), the ~-
globin gene, the Tay-Sachs hexos~m;n;dase gene (HEXA), the
Gaucher gene (GCR), the Canavan aspartoacylase gene (ASPA),
the breast cancer susceptibility gene (BRCAl) and the
Fanconi Anemia Complementation Group C gene (FACC).
PCR amplifications were performed using 1-2 ~g of
genomic DNA or l0 ng of plasmid DNA in l00 ml of reaction
buffer cont~;n;ng l0mM TrisHCl pH 8.3, 50mM KCl, 1.5mM
31
-
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WO97/10366 PCT~S96/14842
MgCl2, 200 mM dNTPs and 0.05-0.l units /~l Taq polymerase
(Perkin-Elmer, Norwalk, CT). For the different disease gene
amplifications, the concentration of primers ranged from
0.2 - 1.6 mM.
For DNA amplifications involving simultaneous
multiplexes of 3 or more amplicons (CFTR 8-plex and 9-plex,
ASPA 3-plex, BRCAl 4-plex, and FACC 3-plex) the primers
were chim~ras of a sequence-specific region with a common
"universal primer sequence" (UPS) as described by Shuber et
al. (33). These primers facilitated rapid multiplex
development and consistently robust amplifications.
DNA amplifications were performed using a Perkin-
Elmer 9600 Thermal Cycler ~Perkin-Elmer, Norwalk, CT). For
CFTR, HEXA, ASPA, BRCAl and FACC, the amplifications were
carried out for 28 cycles with ramping (94C/l0 sec. hold
with 48 sec. ramp, 60C/l0 sec. hold with 36 sec. ramp,
72C/l0 sec. hold with 38 sec. ramp) with a final 74C hold
for 5 minutes before cooling. For ~-globin and GCR, the
amplification program consisted of 28 cycles with a 55C
anneal (94C/l0 sec. hold, 55C/l0 sec. hold, 74C/l0 sec.
hold) with a final 74C hold for 5 minutes before cooling.
Amplification products were analyzed by 2%
agarose gel electrophoresis followed by ethidium bromide
st~;ning and visualization on a W transilluminator
(Fotodyne, New Berlin, WI)
E) Binding of DNA to a Solid Matrix:
8 ~l of the amplified DNA solution prepared as in
D) were added to 50 ~l of a denaturing solution (0.5mM
NaOH, 2.0M NaCl, 25mM EDTA) and spotted onto nylon membrane
32
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WO97/10366 PCT~S96/14842
filters (INC Biotrans). The DNA was then fixed to the
membranes by baking the filters at 80C for 15 minutes under
vacuum.
S Example 2: Hybridization with Allele-Specific
Oligonucleotides (ASOs)
Specific Mutations Examined in the MASDA 106
Hybridization Assay
Mutations from 7 different genes were selected as
candidates for a complex mutation detection assay. The 106
mutations examined included point mutations, deletions and
insertions. Details of the selected mutations and gene
amplifications are listed in Tables 1-8.
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W O 97/10366 PCTAUS96/14842
Table 1. Model System Detailing the Diseases and Mutations Ex:~mine~l using the
MASDA Technology.
Disease Gene Number of Number of Number of
Known ASO Probes PCR Reactions
Mutations
Cystic CFTR > 500 66 2
Fibrosis CF 1 - 31 8-plex
(CF) CF 33 - 68 and
9-plex
globin > 90 14
Th~l~ccerni~
(BT)
Sickle Cell ~-globin 2 2 Same amplicon
(SCA) as
~-Thalassemia
Tay-Sachs Hexos~rnini~ce > 28 3
(TS) A (2-plex)
(HEXA)
Gaucher Glucocerebrosid > 35 8 2
(GCR) ase (3-plex + 1)
(GCR)
Canavan Aspartoacylase ~ 4 4
Disease (ASPA) (3-plex)
(CD)
Breast BRCAl ~ 250 5
Cancer (4-plex)
Susceptibilit
Fanconi Fanconianemia > 8 4
Anemia co~ l.llt;"l~Lion (3-plex)
(FA) C (FACC)
MASDA 106 9
106
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Table 2a: Cystic Fibrosis Mutations Ex~minPd in CFTR 8-plex Amplifications
ExonAmplicon Primers Mutation Mutation
Size (bp) Number Name
12 S 10 16UPCFX12F CF 26 1898+1
16UPCFX12R CF 36 1812-l
CF 37 Y563D
CF 38 PS74H
19 450 UP3CFEX19F CF 23 3849 + 4
UP3CFEX19R CF 27 R1162X
CF 31 3659dC
CF42 R1158X
CF 43 S 1196X
CF 44 I1203V
CF 45 Q1238X
CF 46 3662dA
CF 47 3750dAG
CF 48 3791dC
CF 49 3821dT
9 375 I SUPCFX9F CF 28 A455E
15UPCFX9R
13 335 UP3CFEX 13F CF 29 2184dA
UP3CFEX13R CF 39
CF 40 K710X
3 270 UP3CFEX3F CF 30 G85E
UP3CFEX3R CF33 E60X
CF 34 405 + I
172 UP3CFEXSF CF 25 711 + 1
15UPCFXSR CF 35 G178R
14b lS0 lSUCFX14bF CF 24 2789 + 5
15UCFX 14bR
16 130 16UPCFX16F CF 41 3120 + G
16UPCFX 16R
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Table 2b: Cystic Fibrosis Mutations Exarnined in CFTR 9-plex Amplifications
Exon Arnplicon Primers Mutation Mutation
Size (bp) Number Narne
Int 19 480 15UCFIN19F CF 8 3849 + 10
15UCFIN19R
21 421 REUPCFX21F CF 5 N1303X
15UPCFX21R CF 67 W1310X
CF 68 W1316X
361 UP3CFEX15F CF 60 Q890X
UP3CFEX15R CF 61 2869 + G
CF 62 2909dT
4 307 15UPCFX4F CF6 R117H
15UPCFX4R CF 10 621 + I
CF 21 Y122X
CF 50 444dA
CF 51 556dA
CF 52 574dA
17b 285 L15UCF17BF CF 16 Y1092X
L15UCF17BR CF 64 W1089X
CF 65 3358dAC
7 260 REUPCFX7F CF 12 1078dT
REUPCFX7R CF 17 R347H
CF 18 R347P
CF 20 R334W
CF 53 G330X
CF 54 R352Q
CF 55 S364P
ll 240 15UPCFXllF CF l G542X
15UPCFX1 lR CF 2 G551D
CF 9 R553X
CF 11 1717-1
CF 15 S549R
CF 19 R560T
CF 22 S549N
CF 59 A559T
215 15UPCFXlOF CF 7 DI507
15UPCFX 1 OR CF 13 Q493X
CF 14 V520F
CF 56 508C
CF 57 C524X
CF 58 1677dTA
195 15UPCFX20F CF 3 W1282X
15UPCFX20R CF 4 3905 + T
CF 66 S 1255X
36
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WO 97/10366 PCT/US96/14842
Table 3: ~-th~ semia and Sickle Cell Anemia Mutations Ex~min~l in ,B-globin Gene~mplifir~tions
Exon ~mplir-~n PrimersMutation # Mutation
Size (bp) Name
1-3 1600 GH260 BT 1 IVSl-l
GH283 BT 2 IVS I -6
BT 3 IVS 1-5
BT 4 IVS-110
BT 5 NONS-39
BT 6 IVS2-1
BT 7 IVS-745
BT 8 COD8/9
BT 9 IVS-654
BT 10 41/42
BT l l -29
BT 12 71/72
BT 13 COD24
BT 14 -88
1-3 1600 GH260 SCA I HbS
GH283 SCA 2 HbC
Table 4: Tay-Sachs Mutations Examined in HEXA Gene Amplifications
Exon Amplicon PrimersMutation # Mutation
Size (bp) Name
11/12 530 TSEX1 IF TS 2 Exl l 4 bp
TSEX12R TS 3 Ins
~.,1'~ . 1:
7 190 TSEX7F TS I G269S
TSEX7R
Table 5: Gaucher Mutations F~rnined in GCR Gene Amplifications
Exon Amplicon Primers Mutation # Mutation
Size (bp) Name
10/ll 871 GCRDF GCR5 1448
GCRDR GCR 6 1604
GCR 8
2 358 84IVSF GCR3 84GG
84IVSR GCR 4 IVS2+1
9 319 1226F GCR 1 1297
1226R GCR 2 1226
GCR 7 1342
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Tab1e 6: Canavan Mutations Ex~min~-d in ASPA Gene Amplifications
Exon Amplicon Primers Mutation # Mutation
Size (bp) Name
6 274 CD6F CD 3 E285A
CD6R CD 4 A305E
151 CDSF CD2 Y231X
CDSR
Int2/Ex3 147 CDInt2F CD 1 433-2
CDEx3R
Table 7: Breast Cancer Susceptibility Mutations Examined in BRCAl Amplifications
Exon Amplicon Primers Mutation # Mutation
Size (bp) Name
450 BRCA20F BRC 4 5382+C
BRCA20R
21 315 BRCA21F BRC 5 M1775R
BRCA21R
2 290 BRCA2F BRC 1 185dAG
BRCA2R
270 BRCASF BRC 2 C61G
BRCA5R BRC 3 C64G
Table 8: Fanconi Anemia Mutations F.~min~o.d in FACC Amplifications
Exon Amplicon Primers Mutation # Mutation
Size (bp) Name
366 FAlF FA 2 Q13X
FAlR
6 329 FA6F FA4 R185X
FA6R FA 5 D195V
4 274 FA4F FA 3 IVS4+4
FA4R
38
Oligonycleotide Pools
Allele-specific oligonucleotides (ASOs) were 17-
mers synthesized and HPLC-purified by Operon Technologies
(Alameda, CA). All oligonucleotides were quantitated by
spectrophotometry and tested in independent hybridizations
before being pooled. Specified amounts of individual ASOs
were combined into a pool of 106 ASOs so that the pool
would contain the required amount of each specific ASO
determined to be optimal for the pool hybridization.
Aliquots of pooled ASOs were lyophilized and stored at -
20°C.
Examples of ASOs representing known cystic
fibrosis (CF) mutations are set forth below.
ASO Sequence (17-mer)
.DELTA.F508M 5'ACA/CCA/ATG/ATA/TTT/TC 3' SEQ ID NO:1
G542XM 5'ATT/CCA/CCT/TCT/CAA/AG 3' SEQ ID NO:2
G551DM 5'CTC/GTT/GAT/CTC/CAC/TC 3' SEQ ID NO:3
R553XM 5'CTC/ATT/GAC/CTC/CAC/TC 3' SEQ ID NO:4
W1282XM 5'CTT/TCC/TTC/ACT/GTT/GC 3' SEQ ID NO:5
N1303KM 5'CTT/TCC/TTC/ACT/GTT/GC 3' SEQ ID NO:6
.DELTA.1507M 5'ACA/CCA/AAG/ATA/TTT/TC 3' SEQ ID NO:7
R117HM 5'CGA/TAG/AGT/GTT/CCT/CC 3' SEQ ID NO:8
621+1M 5'GCA/AGG/AAG/TAT/TAA/CT 3' SEQ ID NO:9
S549NM 5'CTC/GTT/GAC/CTC/CAT/TC 3' SEQ ID NO:10
R560TM 5'TAT/TCA/CGT/TGC/TAA/AG 3' SEQ ID NO:11
1717-1M 5'GGA/GAT/GTC/TTA/TTA/CC 3' SEQ ID NO:12
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WO97/10366 PCT~S96/14842
3849+10M 5'ACT/CAC/CAT/TTT/AAT/AC 3' SEQ ID NO:13
3905+TM 5'GTA/GTC/TCA/AAA/AAA/GC 3 SEQ ID NO:14
R347PM 5'GTG/ACC/GCC/ATG/GGC/AG 3 SEQ ID NO:15
1078dTBM 5'CAC/CAC/AAG/AAC/CCT/GA 3 SEQ ID NO:16
2789+5GAM 5'GGA/ATA/TTC/ACT/TTC/CA 3 SEQ ID NO:17
3849+4CM 5'GCA/GTG/TTC/AAA/TCC/CA 3 SEQ ID NO:18
711+1GTM 5'CAT/AAT/TCA/TCA/AAT/TT 3 SEQ ID NO:l9
R1162XM 5'CTC/AGC/TCA/CAG/ATC/GC 3 SEQ ID NO:20
1898+1GAM 5'CAT/ATC/TTT/CAA/ATA/TT 3 SEQ ID NO:21
3659dCM 5'CTT/GTA/GGT/TTA/CCT/TC 3 SEQ ID NO:22
G85EM 5'GAT/TTC/ATA/GAA/CAT/AA 3 SEQ ID NO:23
2184dAM 5'GAT/TGC/TTT/TTG/TTT/CT 3 SEQ ID NO:24
A455EM 5'AAC/CTC/CAA/CAA/CTG/TC 3 SEQ ID NO:25
R334WM 5'TTC/CAG/AGG/ATG/ATT/CC 3 SEQ ID NO:26
Y122XBM 5'AGT/TAA/ATC/GCG/ATA/GA 3 SEQ ID NO:27
S549RBM 5'TCC/CCT/CAG/TGT/GAT/TC 3 SEQ ID NO:28
Q493XM 5'ACT/AAG/AAC/AGA/ATG/AA 3 SEQ ID NO:29
V520FM 5'GAT/GAA/GCT/TCT/GTA/TC 3 SEQ ID NO:30
Y1092XM 5'ACA/GTT/ACA/AGA/ACC/AG 3 SEQ ID NO:31
R347HM 5'GTG/ACC/GCC/ATG/TGC/AG 3 SEQ ID NO:32
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Examples of ASOs representing wild-type or normal
sequences are set forth below.
ASO Seouence (17-mer)
~F508N 5'CAT/AGG/AAA/CAC/CAA/AG 3 SEQ ID NO:33
G542XN 5'ATT/CCA/CCT/TCT/CCA/AG 3 SEQ ID NO:34
G551DN 5'CTC/GTT/GAC/CTC/CAC/TC 3 SEQ ID NO:35
R553XN See G551 DN sequence
W1282XN 5'CTT/TCC/TCC/ACT/GTT/GC 3 SEQ ID NO:36
10 N1303KN 5'TCA/TAG/GGA/TCC/AAG/TT 3 SEQ ID NO:37
~507N 5'ACA/CCA/AAG/ATG/ATA/Tr 3 SEQ ID NO:38
R117HN 5'CGA/TAG/AGC/GTT/CCT/CC 3 SEQ ID NO:39
621+1N 5'GCA/AGG/AAG/TAT/TAC/CT 3' SEQ ID NO:40
S549NN See G551 DN sequence
l5 R560TN 5'TAT/TCA/CCT/TGC/TAA/AG 3 SEQ ID NO:41
1717-lN 5'GGA/GAT/GTC/CTA/TTA/CC 3 SEQ ID NO:42
3849+10N 5'ACT/CGC/CAT/TTT/AAT/AC 3 SEQ ID NO:43
3905+TN 5'GTA/GTC/TCA/AAA/AAG/CT 3 SEQ ID NO:44
R347PN 5'GTG/ACC/GCC/ATG/CGC/AG 3 SEQ ID NO:45
20 1078dTBN 5'CAC/CAC/AAA/GAA/CCC[rG 3 SEQ ID NO:46
2789+5GAN 5'GGA/ATA/CTC/ACT/TTC/CA 3 SEQ ID NO:47
3849+4CN 5'GCA/GTG/TTC/AAA/TCT/CA 3 SEQ ID NO:48
711+1GTN 5'CAT/ACT/TCA/TCA/AAT/TT 3 SEQ ID NO:49
R1162XN 5'CTC/GGC/TCA/CAG/ATC/GC 3' SEQ ID NO:50
25 1898+1GAN 5'CAT/ACC/TTT/CAA/ATA/TT 3' SEQ ID NO:51
3659dCN 5'CTT/GGT/AGG/TTT/ACC/TT 3 SEQ ID NO:52
41
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G85EN 5'GAT/TCC/ATA/GAA/CAT/AA 3' SEQ ID NO:53
2184dAN 5'GAT/TGT/TTT/TTT/GTT/TC 3 SEQ ID NO:54
A455EN 5'AAC/CGC/CAA/CAA/CTG/TC 3' SEQ ID NO:55
R334WN 5'TTC/CGG/AGG/ATG/ATT/CC 3' SEQ ID NO:56
S Y122XBN 5'AGA/TAA/ATC/GCG/ATA/GA 3' SEQ ID NO:57
S549RBN 5'TCC/ACT/CAG/TGT/GAT/TC 3 SEQ ID NO:58
Q493XN 5'ACT/GAG/AAC/AGA/ATG/AA 3 SEQ ID NO:59
V520FN 5'GAT/GAC/GCT/TCT/GTA/TC 3 SEQ ID NO:60
Y1092XN 5'ACA/GGT/ACA/AGA/ACC/AG 3 SEQ ID NO:61
R347HN see R347PN sequence
Dot Blots
Amplified products were denatured using 1.0N
NaOH, 2.OM NaCl, 25mM EDTA pH 8.0, contAining bromophenol
blue (30 ml of 0.1% bromophenol blue/ 10 ml denaturant) for
5 minutes at room temperature. Denatured products were
blotted onto Biotrans membrane (ICN Biomedicals Inc.,
Aurora, OH) using a 96-well format dot blot apparatus (Life
Technologies, Gaithersburg, MD). Membranes were
neutralized in 2x SSC (0.15M NaCl, 0.015M trisodium
citrate) for 5 minutes at room temperature and baked in a
vacuum oven at 80C for 15 minutes. Immediately before use,
the membranes were rinsed in distilled water, and placed in
hybridization solution.
Probe Labeling
Ali~uots of 106 pooled ASOs (representing each
mutation) were thawed, resuspended in distilled water and
end-labeled in a single reaction cont~;n;ng lx kinase
42
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buffer (New England Biolabs, Beverly, MA), 0.135 nmoles of
~-P32-ATP (Dupont, Boston, MA) and 35 units of T4
Polynucleotide kinase (New England Biolabs, Beverly, MA).
The labeling reactions were incubated at 37C for l hour.
The efficiency of the kinase reactions were monitored by
chromatography on cellulose polyethyleneimine (PEI) plates
(J.T. Baker Inc., Phillipsburg, NJ) using 0.75M NaH2PO4 pH
3.5 buffer, followed by exposure of the plates to Kodak X-
Omat X-Ray film (Eastman Kodak Company, Rochester, NY) at
room temperature for 5 minutes.
Hybridizations:
Hybridizations were carried out in plastic bags
cont~in;ng the filters prepared as in Example l above, to
which pooled radiolabelled ASOs were added in a TMAC
hybridization buffer (3.OM TMAC, 0.6% SDS, lmM EDTA, l0mM
sodium phosphate pH 6.8, 5X Denhardt's Solution, and 40
~g/ml yeast RNA).
For this protocol, the 96-well array of spotted
genomic samples was marked with a grid so that positives
identified in the hybridization could be easily located for
the subsequent elution and ASO sequencing. Signal
intensities generated from the different mutation-positive
samples were optimized by adjusting the concentrations of
each mutation-specific oligonucleotide within the
hybridization. In order to achieve uniform hybridization
signals, the final concentration of each labeled mutant ASO
in the pool hybridization ranged from 0.008-l.8 pMol/ml,
with the concentration of cold normal ASOs ranging from 0-
200 fold excess of the corresponding mutant ASO.
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Hybridizations were allowed to proceed overnight
at 52C, with agitation. The membranes were then removed
from the bags and washed for 20 min at room temperature
with wash buffer (3. OM TMAC, O .6% SDS, lmM EDTA, lOmM
sodium phosphate pH 6.8), followed by a second wash in the
same buffer for 20 min at 52C.
Once washed, the blots were wrapped in plastic
wrap and exposed to Kodak X-Omat X-Ray film ~Eastman Kodak
Company, Rochester, NY) at -80C for 15 minutes to l hour.
Example 3: Separation of Hybridized ASOs
Pool-positive samples from hybridizations
performed as in Example 2 are treated as follows: Positive
spots are excised in the form of discs from the nylon
membrane using a st~n~rd single hole paper punch. Each of
the excised membrane discs is then placed in separate .315
ml microcentrifuge tubes containing l00 ~l of sterile
water, and the tubes are incubated at 100C for 15 minutes
(Figure 4.)
Example 4: Design of Complementary Oligonucleotides for
Identification of Bound ASOs
The sequence of the polymers may be determined
directly using chemical sequencing. Alternatively,
polymers may be used in conjunction with complementary
oligonucleotides that contain other sequences in addition
to sequences complementary to the polymers. In these
cases, the polymers serve as primers to form extension
products that contain the additional sequences, and the
extension products are subjected to DNA sequencing.
44
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Specific Mutation Identification
A. By chemical cleavage
The AS0 hybridized to each mutation-positive
sample was identified by eluting and sequencing the AS0.
For the pool of 106 ASOs, sequencing "C" and "G" bases only
was sufficient to unambiguously identify the AS0 sequence
and therefore allowed unequivocal identification of the
corresponding mutation in the DNA sample.
The region of membrane cont~i n; ng each mutation-
positive sample identified in the pool hybridization was
excised and the disc of Biotrans membrane placed in lO0 ml
distilled water and heated at 95C for lO minutes to elute
the bound AS0. After cooling to room temperature, the
membrane disc was discarded, and the eluted ASO was
subjected to chemical sequencing.
Solid-phase chemical cleavage of the ASOs
attached to a solid support was performed according to
Rosenthal et al. (32) with minor changes. This method
permitted simultaneous sequencing of all bound ASOs in a
single reaction vessel. To attach the ASOs to a solid
support prior to chemical cleavage, a small, labeled piece
(6 mm x 3 mm) of CCS paper (32) was immersed in each tube
cont~in;ng eluted ASO, and incubated at 65C for l hour. All
pieces of paper were then combined into a single 50 ml tube
cont~;n;ng 25 ml of distilled water. The papers were then
washed at room temperature 3 times (30 sec./wash) with
distilled water (25 ml/wash) followed by 3 washes (30
sec./wash) with 96% ethanol (25 ml/wash). Papers with
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attached ASOs could be batch washed without cross-
cont~m;n~tion.
Once washed, the papers were air-dried and each
piece cut into two, with 1/3 assigned for the "G~ chemical
cleavage reaction and 2/3 designated for the "C" cleavage
reaction. All "C" reaction ASO-solid supports were combined
into one tube cont~ining 1 ml 4.0M hydroxylamine HCl pH 6.
For "Gn reaction modifications, the combined pieces of
paper were placed in 1 ml 50mM ammonium formate pH 3.5 and
7 ml of DMS added. Reactions were incubated at room
temperature for 10 minutes or 20 minutes for the "G" and
"C" reaction respectively. Batch processing of over 100
sequencing reactions was performed without cross-
contamination of cleavage products.
Washes were performed on the batch of "C"reaction papers and the batch of "G" reaction papers as
described above for washes after attachment of the ASOs to
the solid support. The papers were then air dried and each
piece of paper placed in their designated location in a 96-
well amplification tray (Perkin-Elmer, Norwalk, CT).
To cleave and elute the sequencing products off
the solid support membrane, freshly prepared piperidine (50
~1 of 10% (v/v) piperidine) was added to each well, the
tray was covered with a rubber gasket, and incubated at 90C
for 30 minutes in a therm~1 cycler.
For each cleavage reaction, the piperidine
solution cont~ining the eluted cleavage products was
transferred to a fresh 96-well amplification tray and the
piperidine evaporated. The evaporation step was repeated
twice with 50% ethanol (35 ~l/well). ASO cleavage products
were dissolved in 4 ml loading dye (90% formamide, lx TBE,
46
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0.1~ bromophenol blue, 0.1% xylene cyanol) before gel
electrophoretic resolution. Sequencing gels (20%
polyacrylamide/8M urea/TBE) were pre-run for 1 hour at 2000
V, the samples were loaded, and electrophoresis continued
until the bromophenol blue dye had migrated 14 cm from the
origin. Sequencing gels were exposed to Kodak X-Omat X-ray
film for 24-48 hours.
B. By enzymatic sequencing
Mutation-positive samples were identified, spots
excised and ASOs eluted as described for the chemical
cleavage method. The eluted samples were each placed in
Microcon-10~ concentrators (Amicon Inc., Beverly, MA) and
centrifuged at 14,000 rpm for 15 minutes in a bench top
microfuge. The eluates were then transferred to Microcon-3
concentrators (Amicon Inc., Beverly, MA) and centrifuged at
14,000 rpm for 30 minutes in a bench top microfuge. Both
concentrators were washed twice with 100 ~1 distilled water
per wash. (The Microcon-3 ~ concentrators were washed with
the eluate from the corresponding Microcon-10~
concentrators). The eluted ASOs were recovered from the top
portion of the Microcon-3 ~ concentrator by 3 serial rinses
with 20 ~1 distilled water/rinse, and the fractions pooled.
The samples were lyophilized in a UniVapo~ concentrator
(Integrated Separation Systems, Natick, MA), re-dissolved
in 5~1 of distilled water, and used as sequencing primers
in an enzymatic sequencing protocol designed to identify
the eluted ASO. The sequencing reactions included a pool of
oligonucleotide templates with each template (77-mer)
consisting of a 3' region (17bp) as the primer binding site
uniquely complementary to a specific ASO, and a second
unique region (17bp) consisting of an "ASO-specific
identifier sequencea. Sequencing products were only
observed when an eluted ASO was bound to the complementary
47
CA 0220~234 1997-0~-13
WO 97tlO366 PCT/US96/14842
region of a unique template, acted as a primer and
permitted cycle sequencing to reveal the identity of the
downstream "ASO-specific identifier sequence".
The cycle sequencing reactions contained a pool
of ASO-specific templates (5 fmoles/template), O.5 ~11 of
Thermosequenase buffer concentrate (Amersham Life Science,
Cleveland, OH), O.125 ~l of Thermosequenase (32U/,ul), and
either `G' tPrrnin~tion mix (15mM dATP, 15mM dCTP, 15mM
dTTP, 15mM 7-deaza-dGTP and 4mM ddGTP) or `C' termin~tion
mix (15mM dATP, 15mM dGTP, 15mM dTTP, 15mM 7-deaza-dGTP and
4mM ddCTP) in a reaction volume of 8 ~l. Cycle sequencing
was performed between 95C for 30 seconds and 70C for 1
minute for 30 cycles, followed by a 2 minute incubation at
70C. Sequencing products were resolved on a 15%
acrylamide/7M urea gel before being exposed to Kodak X-Omat
X-ray film at -70C for about 16 hours.
The following are examples of several
complementary oligonucleotides that contain the complement
of the R334W CF mutation-specific ASO identified
hereinabove.
Version 1: ASO as sequencing primer
SEQ ID NO:62
3'-AAGGTCTCCTACTAAGG-TCTCGCTTCGTTTCATCTCATCTCG-5'
ASO complement "Tag"
In this embodiment, an ASO is incubated with the
complementary oligonucleotide in a Sanger sequencing
reaction, and the sequence is determined directly.
48
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Version 2: Cycle sequencing of eluted ASO
3'-AAGGTCTCCTACTAAGG-TCTCGCTTCGTTTCATCTCATCTCG-
ASO complement "Tag"
ATCGATCGATCGATCGATCGATCG-5' SEQ ID NO:63
Universal Primer Sequences
In this embodiment, an ASO serves as a primer for
a single extension reaction. The extension product is then
subjected to cycle sequencing, using the universal primer
to prime the sequencing reaction (see Example 5 below.).
Version 3: Amplification of complementary oligonucleotide
for Sanger sequencing
3'-AAGGTCTCCTACTAAGG-CGCCAGGGllllCCCAGTCA-
ASO complement "sequencing target"
TCTCGCTTCGTTCATCTCATCTCG-ATCGATCGATCGATCGATCGATCGA-5'
"Tag" Universal Primer Sequence
SEQ ID NO:64
In this embodiment, an ASO serves as a primer for
a single extension reaction. The extension product is then
amplified using the universal primer sequence and the ASO
as amplification primers. Finally, the amplification
products are subjected to Sanger sequencing using as a
primer an oligonucleotide corresponding to the sequencing
target (see Example 6 below.).
49
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W097tlO366 PCTtUS96tl4842
Example 5: Cycle Sequencing of ASOs
A) Extension Reaction
A separated mutation-specific oligonucleotide,
designated R334W and having the sequence
5'-TTCCAGAGGATGATTCC-3' SEQ ID NO:65 is added to a
reaction mix cont~;ning reaction components necessary for a
single round of extension. The complementary
oligonucleotide (Version 2 in Example 4 above) contains a
universal primer sequence at its 5' end, separated by 25-30
bases from the complement to R334W at its 3' end. The
extension reaction contains the following components:
25 ~1 separated ASO
5 ~1 lOX buffer (0.5mM Tris-HC1 pH 7.5, O.lM
MgCl2, lOmM dithiothreitol)
1 ~1 dNTPs (2.5 mM each)
1 ~l complementary oligonucleotides (100 ng/ml)
13 ~1 H20
1 ~1 Klenow fragment of DNA polymerase (lOU/~1)
The reaction is allowed to proceed at room temperature for
30 minutes.
B) Cycle Sequencing:
An aliquot of the above reaction is added to a
PCR reaction mix cont~;n;ng two or more dideoxynucleotide
analogues (ddNTPs), according to the following protocol:
10 ~l extension products
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WO97/10366 PCT~S96/14842
5 ~1 lOX buffer (300 mM Tris-HCl pH 9.0, 50 mM
MgCl2, 300 mM KCl)
5 ~1 universal primer (1 pmole)
10 ~1 2mM ddATP, ddCTP, ddGTP; 100 ~M dATP, dCTP,
dGTP, dTTP
19 ~ll H20
1 ~1 Taq polymerase (lOU/~l~
30 cycles of amplification are performed, creating a
heterogeneous population of random t~rmin~tion products
that termin~te at positions corresponding to nucleotides
downstream of the universal primer sequence. The products
of the PCR reaction are then separated in a denaturing
polyacrylamide gel, creating a h~n~;ng pattern specific for
this ASO. The electrophoretic pattern is analyzed by
autoradiography or fluorimetry.
Example 6: Amplification and Sequencing of Complementary
Oligonucleotides
A separated mutation-specific oligonucleotide,
designated R334W and having the sequence
5'-TTCCAGAGGATGATTCC-3' is added to a reaction mix
cont~ining reaction components for extension as in Example
5, Step A. The complementary oligonucleotide (Version 3 in
Example 4 above) contains a universal primer sequence at
its 5' end, a "tag" sequence, "sequencing target~ sequence,
followed by the complement to R334W at its 3' end.
Following the extension reaction, an aliquot of the
reaction is added to an amplification mixture cont~;n;ng
the following components:
CA 0220~234 1997-0~-13
WO97/10366 PCT~S96/14842
3 ~l extension products
l ~l universal amplification primer (lO~M)
2.5 ~l dATP, dTTP, dCTP, dGTP (2mM each)
2 ~l 40 mM MgCl2
5 ~l lO0 mM Tris-HCl pH 8.3, 500 mM KCl
26.4 ~l H2O
O.l ~l Amphitaq DNA polymerase (5 U/~l).
The reaction is then subjected to 35 cycles of
amplification, using a GeneAmp PCR System 9600
Thermocycler. 2 ~l of the amplification products are then
removed and subjected to Sanger sequencing, using the
Sanger sequencing primer.
Example 7: RNA as a Target Nucleic Acid
In a similar manner to Example l, which describes
DNA as a target nucleic acid, RNA may also be used as a
target nucleic acid.
A) Preparation of RNA from Target Cells
Cells are collected by centrifugation, and the
supernatant is removed. The cell pellet is suspended in
cold lysis buffer (140 mM NaCl, 1.5 mM MgCl2, l.0 mM Tris-
Cl, pH 8.5, 0.5% NP-40, and Rnasin~ (Promega, Inc.)).
Cellular debris is pelleted by centrifugation for 5 minutes
at 4C at 5000 x g. The supernatant is transferred to a
fresh tube and the EDTA concentration brought to lO mM.
Proteins are removed by extraction with phenol-chloroform
saturated with aqueous lO mM Tris, pH 8.5. The aqueous
phase is precipitated with sodium acetate at pH 5.2 and 2.5
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WO97/10366 PCT~S96/14842
volumes of ice cold ethanol overnight at 10C RNA is
collected by centrifugation at lO,000 x g at 4C for 30
minutes.
B) Conversion of RNA to cDNA Before Amplification
RNA may be used directly in the manner of the
present invention, or converted to amplified DNA via a
reverse transcription PCR protocol. According to this
protocol, l ~g of RNA is mixed with lO0 pmol of appropriate
primers, lmM dNTPs, lU/~l RNasin~ in 20 ~l PCR buffer (50
mM KCl, 20 mM Tris, pH 8.4, 2.5 mM MgCl2) and 200 U of
reverse transcriptase. The mixture is incubated at 23C for
lO minutes, then 42C for 45 minutes, then 95C for 5
minutes, and then quick chilled. Conventional PCR
Protocols, similar to those described in Example l, may be
used to amplify the resultant cDNA.
Example 8: Unique Probe Identifiers
Instead of det~rm;ning the identity of the
separated polymer using chemical or enzymatic sequencing
reactions, it is also possible to label each probe polymer
with a unique identifier moiety that can be detected
directly or indirectly. The following description merely
onctrates examples of the full range of unique probe
identifiers that one of skill would readily understand to
have use in the present invention.
CA 0220~234 1997-0~-13
W097tlO366 PCT~S96/14842
A) Fluorescent Labels
Oligonucleotides are hybridized to immobilized
nucleic acid targets in a similar manner as described in
Example 2 above, except that each ASO in the pool is
labeled with a unique fluorescent probe instead of 32p . For
example, ASOs designated F508M, G542XM, G55IDM and R553XM
are labeled with Texas Red, tetramethylrhodamine,
fluorescein, and Cy3, respectively. Similar to Example 3,
bound ASOs can be detected as having been bound prior to
separation. In this Example, ASO bin~lng is detected by
fluorescence of the conjugated label either visually or by
any number of automated methods. After separation, the ASO
can be positively identified by measuring emission
wavelength in response to fluor excitation.
B) Molecular Weight Labels
Oligonucleotides are hybridized to immobilized
nucleic acid targets in a similar manner as described in
Example 2 above, except that each ASO in the pool is
additionally labeled with a unique molecular weight
modifying entity. For example, the four ASOs described in
Example 8A are each derivatized with a 5' oligomeric
hexaethyleneoxide (HEO) tail of differing length. ASOs
designated ~F508M, G542XM, G551DM and R553XM can be labeled
with lengths of 5, lO, 15 and 20 HEO units, respectively.
The tails are added using st~n~rd DNA synthesis protocols
such as those described in Nucleic Acid Res, 22: 4527. The
HEO tail does not participate in hydrogen bonding but does
give a unique molecular weight to each ASO. The ASO can be
identified without further modification by distinguishing
the separated ASOs by molecular weight, using any number of
54
CA 0220~234 1997-0~-13
WO97/10366 PCT~S96/14842
commonly recognized methods, such as gel or capillary
electrophoresis.
C) Alternative Molecular Weight Labeling Method
An additional method of utilizing molecular
weight identification of the hybridizing polymer is to add
an additional number of nucleotides to the polymer
enzymatically after separation from target nucleic acid.
In a preferred embodiment of this method, the separated
polymer, after hybridization to the immobilized nucleic
acid target, is collected into a tube cont~ining
oligonucleotides, each of which is complementary to one
member of the polymer pool used to probe the target nucleic
acid. In addition to a portion that is complementary to
the polymer, the oligonucleotide also contains an
additional sequence, the length of which is unique for that
oligonucleotide. When the polymer and oligonucleotide
hybridize, the polymer can subsequently be used as a primer
to enzymatically extend the polymer to the full length of
the complementary oligonucleotide. During this process, a
direct or indirect label, as described above, may be
incorporated. The extended oligonucleotide can be
identified by det~rmining the relative molecular weight of
the labeled product by any number of established methods,
such as gel or capillary electrophoresis.
Example 9: Ligation of Polymers
Ligation based techniques are known for
identifying polymers probes that have perfectly hybridized
to a sample. Ligation is often used in such techniques to
- distinguish perfect from imperfect hybridization at the
junction of adjacent polymer probes. This is particularly
35 usefui for det~rm;n;ng genetic alterations. (Landegren et
CA 0220~234 lgg7-0~-l3
W097/10366 PCT~S96/14842
al., 1988, Science 241:1078). LCR is one technique that
results in amplification of the ligated products and can be
used to aid in obt~ining sufficient copies of the product
to determine the presence of the target sequence. Gap-LCR
is a modification of LCR that reduces the background
generated by target-independent ligation. Other ligation
related amplification techniques are listed above. Figure
5 schematically depicts some examples of ligation
techniques that can be used in the present invention. In
these schemes polymers form ligation probes that, e.g.,
flank the site of a genetic alteration. One of the
polymers (or probes) of each pair has a capture molecule
attached. The other has a reporter molecule attached.
After the polymers are hybridized to immobilized samples,
and ligated, any non-hybridized polymers may be washed
away. Washing away of unhybridized polymers is not always
necessary, although it is strongly preferred, particularly
where a large number of polymers is reacted with the
samples (an excess of unhybridized polymers can interfere
with identification of ligated polymers by e.g.
sequencing). Nor does the sample need to be immobilized
since it is possible to carry out hybridization and
ligation of the probes in solution. Furthermore, imperfect
hybrids can form under conditions used in ligation
procedures. TMAC and strict temperature control are often
not required. Imperfect hybrids, however, do not register
as false positives because they do not ligate, and
therefore are not identified.
The ligated products can then be amplified to
allow easier identification. For example, LCR
thermocycling accomplishes this. Alternately, it is
possible to amplify the ligated products using other
techniques, such as PCR. It is also possible to amplify
the amount of sample DNA prior to the hybridization step so
56
CA 0220~234 lgg7-o~-l3
W097/10366 PCT~S96/14842
that, if a sufficient quantity of polymer probes is used,
the ligated products will not need to be amplified.
The hybridized and ligated polymers are then
captured on a solid support. The presence of the reporter
molecule is then determined. If the reporter is present,
then ligation has occurred, and the presence of the target
molecule determined.
Various schemes to identify the target sequence
that the ligated polymers are specific for will be apparent
to one skilled in the art. Four such schemes are depicted
in Figures 6-9, indicated as "Models" 1-4. In Model l
~Figure 6), genetic alterations, target nucleic acid
sequences, or randomly permuted alterations that resulted
in ligation of the polymers are identified through chemical
cleavage sequencing of the products, i.e., Maxam-Gilbert
type sequencing. In Model 2 (Figure 7), the ligation
products are sequenced in a conventional Sanger type
sequencing reaction. Specifically, after the ligation
products are captured on a solid phase, a heterogeneous
population of sequences is added that includes a sequence
that hybridizes to the ligation product. Sequencing is
carried out, and the ligation product identified. In Model
3, (Figure 8) ligation products are directly sequenced by
conventional Sanger sequencing using heterogeneous primers
that are complementary to sequences in the "B" portion of
the ligation product depicted. Alternately, sequencing is
done using a primer complementary to a common 3' tether
added to the "B" probes. In Model 4 (Figure 9), the
ligation product is used as a template for a linear
amplification using a universal priming sequence. The
reaction can be performed with the product either attached
to a solid phase or in solution. The products are
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WO97/10366 PCT~S96/14842
conventionally sequenced by Sanger sequencing using a
primer complementary to the tether sequence.
Example lO: ARMS Amplification of Polymers
The amplification refractory mutation system is a
known PCR type system for detPrmin;ng mutations and can be
used to practice the present invention. For example,
polymers are synthesized that are complementary to a
sequence that may contain a mutation, and that act as
primers when hybridized to the target DNA. Primers that
are complementary to wild type sequence(s) are unlabelled.
Primers that are complementary to a mutant sequence(s) are
labelled. A second polymer is synthesized that is designed
to act as a primer allowing PCR amplification when used in
combination with the first set of primers. The second
polymer is attached to one member of a binding pair, e.g.,
biotin, that allows capture of amplified PCR products on a
solid phase. The polymer/primers are hybridized to the
sample, and PCR thermocycling is then carried out. The
amplified products are then exposed to a solid phase having
a b;n~;ng partner (e.g. avidin) attached to its surface.
Presence of a signal on the amplified products bound to the
support surface indicates that a mutant sequence was
present in the sample. The bound products can then be
identified using methods described above, e.g. Sanger
sequencing.
Invention methodologies provided herein retain
the capacity for large sample throughput while reducing the
number of hybridizations involved in performing multiple
mutation analysis.. If the number of probes for any
diagnostic test is extremely large, the large number of
independent hybridizations to be performed on pool positive
samples reduces the cost effectiveness of the invention
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CA 0220~234 1997-0~-13
WO97/10366 PCT~S96/14842
methodology. Using the MASDA approach of the instant
invention, the disadvantages of both individual sample
hybridizations and independent probe hybridizations are
avoided. By eluting and interrogating the sequence of the
mutation-specific oligonucleotides hybridized to a DNA
sam.ple, MASDA eliminates the need for secondary independent
hybridizations. Therefore, in a single day, hundreds of
different samples can be simultaneously analyzed in a
single hybridization cont~; n i ng a complex mixture of
hundreds of mutation-specific oligonucleotides. Also, by
generating short and unique band patterns for the
hybridized and eluted oligonucleotides, multiple samples
can be analyzed by stagger loading samples across multiple
lanes of a gel. Therefore, using currently available
automated sequencers, specific mutation identification can
easily be performed on hundreds of pool positive samples at
a rate in excess of lS0 samples/hour.
In addition to high throughput sample capacity
and complex mutation analysis, there are several other
advantages to the invention MASDA technology. MASDA is an
extremely flexible, modular platform. This is very
important in a field such as genetic diagnostics, where the
number of relevant genes, and mutations identified in each
gene, change rapidly. Having oligonucleotide probes in
solution, it is possible to mix and match probes on ~m~n~,
therefore allowing clinical laboratories to cost-
effectively customize diagnostic assays. There is also
flexibility in sample preparation, and target detection.
The sample nucleic acid can be either DNA or RNA.
For exemplification of multiple target
amplifications, PCR was utilized as the amplification
procedure. However, the present invention, MASDA, is
compatible with any amplification technology, and does not
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CA 0220~234 lgg7-o~-l3
WO97/10366 PCT~S96/14842
require any processing of amplification products prior to
mutation detection and identification. This becomes a very
important issue when large numbers of samples need to be
analyzed in a single assay. Since the sample nucleic acid
does not need to be fragmented, long PCR products can be
analyzed, as well as the multiplex amplicons demons~rated
in this study.
Currently, significant efforts are being
established to develop informative databases on
genotype/phenotype associations of existing and new
mutations within known disease genes. Additionally, there
is an ever increasing interest in establishing the
relationships between genotypes of patients involved in
clinical trials and their response to various therapeutic
treatments. The preesnt invention MASDA will facilitate
the development of oligonucleotide libraries representative
of previously identified expressed sequence tags or bi-
allelic markers identified within the human genome.
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