Polymorphism analysis by nucleic acid structure probing
First Claim
Patent Images
1. A method, comprising:
- a) providing;
i) one or more copies of a target DNA comprising a folded structure comprising one or more double stranded regions and one or more single stranded regions; and
ii) a plurality of different oligonucleotide probes complementary to at least a portion of said target DNA;
b) mixing said one or more copies of target DNA and said plurality of oligonucleotide probes under conditions such that at least two of said probes hybridize to at least one copy of said target DNA to form probe/folded target complexes;
c) detecting said probe/folded target complexes; and
d) generating a fingerprint of said folded structure, wherein said generating comprises measuring the level of complex formation between said target nucleic acid and individual members of said plurality of oligonucleotide probes.
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Abstract
The present invention relates to methods and compositions for analyzing nucleic acids. In particular, the present invention provides methods and compositions for the detection and characterization of nucleic acid sequences and sequence changes. The methods of the present invention permit the detection and/or identification of genetic polymorphism such as those associated with human disease and permit the identification of pathogens (e.g., viral and bacterial strain identification).
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Citations
26 Claims
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1. A method, comprising:
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a) providing;
i) one or more copies of a target DNA comprising a folded structure comprising one or more double stranded regions and one or more single stranded regions; and
ii) a plurality of different oligonucleotide probes complementary to at least a portion of said target DNA;
b) mixing said one or more copies of target DNA and said plurality of oligonucleotide probes under conditions such that at least two of said probes hybridize to at least one copy of said target DNA to form probe/folded target complexes;
c) detecting said probe/folded target complexes; and
d) generating a fingerprint of said folded structure, wherein said generating comprises measuring the level of complex formation between said target nucleic acid and individual members of said plurality of oligonucleotide probes. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method, comprising:
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a) providing;
i) two or more copies of a first target DNA, said first target DNA comprising
1) a sequence comprising a folded structure, said folded structure comprising one or more double stranded regions and one or more single stranded regions; and
2) first and second portions,ii) two or more copies of a second target DNA, said second target DNA comprising
1) a sequence comprising a folded structure, said folded structure comprising one or more double stranded regions and one or more single stranded regions; and
2) first and second portions, wherein said first portion of said second target DNA is identical in sequence to said first portion of said first target DNA, and wherein said second portion of said second target DNA differs in folded structure from said second portion of said first target DNA because of a variation in nucleic acid sequence of said second target DNA relative to said sequence of said first target DNA;
iii) two or more copies of a first oligonucleotide probe and two or more copies of a second oligonucleotide probe, said first oligonucleotide probe complementary to at least a portion of said first portion of said first and second target DNAs and said second oligonucleotide probe complementary to at least a portion of said second portion of said first and second target DNAs; and
iv) a solid support comprising first, second, third and fourth testing zones, each zone capable of capturing and immobilizing said first and second oligonucleotide probes;
b) contacting at least one copy of said first target DNA with at least one copy of said first oligonucleotide probe under conditions such that said first probe binds to said first target DNA to form a probe/folded target complex in a first mixture;
c) contacting at least one copy of said first target DNA with at least one copy of said second oligonucleotide probe under conditions such that said second probe binds to said first target DNA to form a probe/folded target complex in a second mixture;
d) contacting at least one copy of said second target DNA with said first oligonucleotide probe under the conditions used for said first mixture, to form a third mixture;
e) contacting at least one copy of said second target DNA with said second oligonucleotide probe under conditions used for said second mixture, to form a fourth mixture;
f) adding said first, second, third and fourth mixtures to said first, second, third and fourth testing zones of said solid support, respectively, under conditions such that said probes are captured and immobilized producing captured probe/folded target duplexes; and
g) detecting said captured probe/folded target duplexes. - View Dependent Claims (14, 15, 16, 21)
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17. A method, comprising:
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a) providing;
i) a first folded target DNA comprising
1) one or more double stranded regions and one or more single stranded regions and
2) first and second portions;
ii) a second folded target DNA having a nucleic acid sequence comprising
1) one or more double stranded regions and one or more single stranded regions,
2) a first portion that is identical in sequence to said first portion of said first folded target, and
3) a second portion that differs from said second portion of said first folded target because of a variation in nucleic acid sequence relative to said first folded target;
iii) two or more copies of a first oligonucleotide probe and two or more copies of a second oligonucleotide probe, said first oligonucleotide probe complementary to at least a portion of said first portion of said first and second folded targets and said second oligonucleotide probe complementary to at least a portion of said second portion of said first and second folded targets; and
b) contacting said first folded target with at least one copy of each of said first and second oligonucleotide probes to form probe/first-folded target complexes;
c) contacting said second folded target with at least one copy of each of said first and second oligonucleotide probes to form probe/second-folded target complexes;
d) detecting said probe/first-folded target complexes and probe/second-folded target complexes; and
e) comparing the levels of said probe/first-folded target complexes and probe/second-folded target complexes. - View Dependent Claims (18, 19, 20)
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22. A method comprising:
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a) providing;
i) a first target DNA comprising
1) a nucleic acid sequence comprising a folded structures said folded structure comprising one or more double stranded regions and one or more single stranded regions; and
2) first and second portions,ii) a second target DNA comprising
1) a nucleic acid sequence comprising a folded structure, said folded structure comprising one or more double stranded regions and one or more single stranded regions; and
2) first and second portions, wherein said first portion of said second target DNA is identical in sequence to said first portion of said first target DNA, and wherein said second portion of said second target DNA differs in folded structure from said second portion of said first target DNA because of a variation in nucleic acid sequence of said second target DNA relative to said sequence of said first target DNA;
iii) two or more copies of an oligonucleotide probe, wherein said probe is complementary to at least a portion of said first portion of said first and second folded DNA targets; and
iv) a solid support comprising first and second testing zones, each zone capable of capturing and immobilizing said oligonucleotide probe;
b) contacting said first target DNA with said first oligonucleotide probe under conditions such that said oligonucleotide probe binds to said first target DNA to form a probe/folded target complex in a first mixture;
c) contacting said second target DNA with said oligonucleotide probe under the conditions used for said first mixture, to form a second mixture;
d) adding said first and second mixtures to said first and second testing zones of said solid support, respectively, under conditions such that said probes are captured and immobilized, producing captured probe/folded target duplexes; and
e) detecting said captured probe/folded target duplexes in each testing zone. - View Dependent Claims (23, 24, 25, 26)
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Specification