Direct sequence identification of mutations by cleavage- and ligation-associated mutation-specific sequencing
First Claim
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1. A method for identifying one or more genetic alterations in a target sequence present in a DNA sample, which comprises:
- a) immobilizing a first DNA sample, said first DNA having a 5'"'"' end and a 3'"'"' end on a solid support under conditions such that said 5'"'"' end is bound to said support and said 3'"'"' end is unbound;
b) hybridizing said immobilized sample with a second DNA having a 5'"'"' end and a 3'"'"' end wherein said second DNA does not contain the alteration(s), to form heteroduplex DNA containing a mismatch region at the site of an alteration(s);
c) cleaving one or both strands of said heteroduplex adjacent to said mismatch region to form a gap at or in the vicinity of said alteration;
d) subjecting said cleaved heteroduplex to conditions of denaturation to dissociate said second DNA and cleaved first DNA 3'"'"' to the site of cleavage from immobilized remaining first DNA;
e) removing DNA strands cleaved in step c and dissociated in step d from said immobilized remaining first DNA;
f) ligating a single-stranded oligonucleotide primer of known sequence to the unbound end of said immobilized remaining first DNA to form a ligation product;
(g) treating said ligation product with a DNA polymerase and an oligonucleotide complementary to said primer of known sequence in the presence of dideoxynucleotides or four nucleotide triphosphates and determining the nucleotide sequence adjacent to the ligated primer sequence; and
h) comparing said nucleotide sequence with a predetermined cognate wild-type sequence to identify said genetic alteration(s).
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Abstract
The present invention provides methods for identifying the presence, location and sequence of one or more genetic alterations in one or more DNA molecules. Further provided are methods for positional cloning of a gene of interest.
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Citations
47 Claims
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1. A method for identifying one or more genetic alterations in a target sequence present in a DNA sample, which comprises:
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a) immobilizing a first DNA sample, said first DNA having a 5'"'"' end and a 3'"'"' end on a solid support under conditions such that said 5'"'"' end is bound to said support and said 3'"'"' end is unbound; b) hybridizing said immobilized sample with a second DNA having a 5'"'"' end and a 3'"'"' end wherein said second DNA does not contain the alteration(s), to form heteroduplex DNA containing a mismatch region at the site of an alteration(s); c) cleaving one or both strands of said heteroduplex adjacent to said mismatch region to form a gap at or in the vicinity of said alteration; d) subjecting said cleaved heteroduplex to conditions of denaturation to dissociate said second DNA and cleaved first DNA 3'"'"' to the site of cleavage from immobilized remaining first DNA; e) removing DNA strands cleaved in step c and dissociated in step d from said immobilized remaining first DNA; f) ligating a single-stranded oligonucleotide primer of known sequence to the unbound end of said immobilized remaining first DNA to form a ligation product; (g) treating said ligation product with a DNA polymerase and an oligonucleotide complementary to said primer of known sequence in the presence of dideoxynucleotides or four nucleotide triphosphates and determining the nucleotide sequence adjacent to the ligated primer sequence; and h) comparing said nucleotide sequence with a predetermined cognate wild-type sequence to identify said genetic alteration(s). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24)
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21. A method for identifying one or more genetic alterations in a target sequence present in a genomic DNA sample, which comprises:
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a) immobilizing a first DNA sample, said first DNA having a 5'"'"' end and a 3'"'"' end on a solid support under conditions such that said 5'"'"' end is bound to said support and said 3'"'"' end is unbound; b) hybridizing said immobilized DNA sample with a second DNA sample, said second DNA having a 5'"'"' end and a 3'"'"' end, and wherein said second sample does not contain the alteration(s), to form heteroduplex DNA containing a mismatch region at the site of an alteration(s); c) treating said heteroduplex with terminal transferase in the presence of a dideoxynucleotide to block unbound ends thereof; d) contacting said heteroduplex with bacteriophage T4 endonuclease 7 to cleave one or both strands of said heteroduplex adjacent to said mismatch region to form a gap at or in the vicinity of said alteration; e) subjecting said cleaved heteroduplex to conditions of denaturation to dissociate said second DNA and cleaved first DNA 3'"'"' to the site of cleavage from immobilized remaining first DNA; f) removing DNA strands cleaved in step d and dissociated in step (e) from said immobilized remaining first DNA; g) ligating a single-stranded oligonucleotide primer having the sequence 5'"'"'-CAGTAGTACAACTGACCCTTTTGGGACCGC-3'"'"' (SEQ ID NO;
1) to the unbound end of immobilized remaining first DNA to form a ligation product;(h) treating said ligation product with a DNA polymerase and an oligonucleotide complementary to said primer of known sequence in the presence of dideoxynucleotides or four nucleotide triphosphates and determining the nucleotide sequence adjacent to ligated primer sequence; and i) comparing said nucleotide sequence with a predetermined cognate wild-type sequence to identify said alteration(s). - View Dependent Claims (25)
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22. A method for identification of one or more mutation(s) in a DNA, which comprises:
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a) immobilizing one or more first DNA samples, said first DNA having a respective 5'"'"' end and a respective 3'"'"' end, on a solid support under conditions such that said 5'"'"' end is bound to said support and said 3'"'"' and is unbound; b) hybridizing said immobilized sample(s) with a second DNA sample, said second DNA having a respective 5'"'"' end and a respective 3'"'"' end, and wherein said second sample does not contain the mutation(s), to form heteroduplex DNA containing a mismatch region at the site of a mutation; c) chemically blocking unbound ends on said heteroduplex DNA; d) treating said heteroduplex DNA so that one or both strands are cleaved within or adjacent to said mismatch region to form a gap at or in the vicinity of said alteration; e) subjecting said cleaved heteroduplex to conditions of denaturation to dissociate said second DNA and cleaved first DNA 3'"'"' to the site of cleavage from immobilized remaining first DNA; f) removing DNA strands cleaved in step (d) and dissociated in step (e) from immobilized remaining first DNA; g) ligating a single-stranded oligonucleotide primer of known sequence to the unbound end of said immobilized remaining first DNA to form a ligation product; (h) treating said ligation product with a DNA polymerase and an oligonucleotide complementary to said primer of known sequence in the presence of dideoxynucleotides or four nucleotide triphosphates and determining the nucleotide sequence adjacent to ligated primer sequence; and i) comparing said nucleotide sequence with one or more predetermined cognate wild-type sequences to identify said mutation(s). - View Dependent Claims (26)
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23. A method for multiplex identification of one or more mutations in a DNA, the method comprising:
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a) obtaining one or more first DNA samples, said DNA having a respective 5'"'"' end and a respective 3'"'"' end; b) amplifying one or more target sequences in each of said samples; c) immobilizing said amplified sequences on a solid support under conditions such that said 5'"'"' end is bound to said support and said 3'"'"' end is unbound; d) hybridizing said immobilized sample(s) with a second DNA sample, said second DNA having a respective 5'"'"' end and a respective 3'"'"' end, and wherein said second sample does not contain the mutation(s), to form heteroduplex DNA containing a mismatch region at the site of a mutation; e) chemically blocking unbound ends on said heteroduplex DNA; f) treating said heteroduplex DNA so that one or both strands are cleaved within or adjacent to said mismatch region to form a gap at or in the vicinity of said mutation; g) subjecting said cleaved heteroduplex to conditions of denaturation to dissociate said second DNA and cleaved first DNA 3'"'"' to site of cleavage from immobilized remaining first DNA; h) removing DNA strands cleaved in step (f) and dissociated in step (g) from said immobilized remaining first DNA; i) ligating a single-stranded oligonucleotide primer of known sequence to the unbound end of said immobilized remaining first DNA to form a ligation product; (j) treating said ligation product with a DNA polymerase and an oligonucleotide complementary to said primer of known sequence in the presence of dideoxynucleotides or four nucleotide triphosphates and determining the nucleotide sequence adjacent to ligated primer sequence; and k) comparing said nucleotide sequence with one or more predetermined cognate wild-type sequences to identify said mutation(s). - View Dependent Claims (27)
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28. A method for positional cloning of a gene of interest, the method comprising:
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a) immobilizing a first DNA sample from an individual displaying a given phenotype, said first DNA having a 5'"'"' end and a 3'"'"' end on a solid support under conditions such that said 5'"'"' end is bound to said support and said 3'"'"' end unbound; b) hybridizing said immobilized sample with a second DNA sample, said second DNA having a 5'"'"' end and a 3'"'"' end, and wherein said second DNA sample is from one or more individual(s) not displaying said phenotype to form heteroduplex DNA containing a mismatch region at the site of a genetic alteration; c) cleaving one or both strands of said heteroduplex DNA to form a gap at or in the vicinity of said alteration; d) subjecting said cleaved heteroduplex to conditions of denaturation to dissociate said second DNA from said first DNA; e) removing DNA strands cleaved in step (c) and dissociated in step (d) from said first DNA; f) ligating a single-stranded oligonucleotide primer of known sequence to the 3'"'"' end of first cleaved DNA to form a ligation product; g) treating said ligation product with a DNA polymerase and an oligonucleotide complementary to said primer of known sequence in the presence of dideoxynucleotides or four nucleotide triphosphates and determining the nucleotide sequence adjacent to ligated primer sequence; h) preparing a synthetic oligonucleotide comprising all or part of said determined nucleotide sequence; and i) identifying a DNA clone that hybridizes to said oligonucleotide. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47)
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Specification