Random array DNA analysis by hybridization
First Claim
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1. A method for determining all or part of the sequence of a plurality of target nucleic acid sequences comprising:
- (a) providing an array comprising a plurality of different target nucleic acid sequences randomly distributed on a surface of said array, wherein each target nucleic acid comprises a first target domain and an adjacent second target domain, wherein said first and second target domains each comprise multiple nucleotides;
(b) contacting said array with;
(i) a first probe set comprising;
1. a first probe pool comprising probes complementary to said first target domains;
2. a second probe pool comprising probes complementary to said second target domains;
wherein at least one of said first and second probe pools comprises a label;
(c) ligating probes from said first probe pool and probes from said second probe pool when hybridized to said first and second target domains to form first ligated probes;
(d) detecting said first ligated probes to determine at least one nucleotide of said first target domain;
(e) removing said first ligated probes;
(f) repeating steps (b)-(d) to determine all nucleotides of said first target domain, wherein each nucleotide of said first target domain is determined multiple times.
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Abstract
The invention relates to methods and devices for analyzing single molecules, i.e. nucleic acids. Such single molecules may be derived from natural samples, such as cells, tissues, soil, air and water without separating or enriching individual components. In certain aspects of the invention, the methods and devices are useful in performing nucleic acid sequence analysis by probe hybridization.
107 Citations
26 Claims
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1. A method for determining all or part of the sequence of a plurality of target nucleic acid sequences comprising:
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(a) providing an array comprising a plurality of different target nucleic acid sequences randomly distributed on a surface of said array, wherein each target nucleic acid comprises a first target domain and an adjacent second target domain, wherein said first and second target domains each comprise multiple nucleotides; (b) contacting said array with; (i) a first probe set comprising; 1. a first probe pool comprising probes complementary to said first target domains; 2. a second probe pool comprising probes complementary to said second target domains; wherein at least one of said first and second probe pools comprises a label; (c) ligating probes from said first probe pool and probes from said second probe pool when hybridized to said first and second target domains to form first ligated probes; (d) detecting said first ligated probes to determine at least one nucleotide of said first target domain; (e) removing said first ligated probes; (f) repeating steps (b)-(d) to determine all nucleotides of said first target domain, wherein each nucleotide of said first target domain is determined multiple times. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A method for determining all or part of the sequence of a plurality of target nucleic acid sequences comprising:
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(a) providing an array comprising a plurality of different target nucleic acid sequences randomly distributed on a surface of said array, wherein each target nucleic acid comprises a first target domain and an adjacent second target domain, wherein said first and second target domains each comprise multiple nucleotides; (b) contacting said array with; (i) a first probe set comprising; 1. a first probe pool comprising probes complementary to said first target domains; 2. a second probe pool comprising probes complementary to said second target domains; wherein at least one of said first and second probe pools comprises a label; (c) ligating probes from said first probe pool and probes from said second probe pool when hybridized to said first and second target domains to form first ligated probes; (d) detecting said first ligated probes to determine at least one nucleotide of said first target domain; (e) removing said first ligated probes; (f) contacting said array with; (i) a second probe set comprising; 1. a third probe pool comprising probes complementary to said first target domains; 2. a fourth probe pool comprising probes complementary to said second target domains; wherein at least one of said third and fourth probe pools comprises a label; (g) ligating probes from said third probe pool and probes from said fourth probe pool when hybridized to said first and second target domains to form second ligated probes; (h) detecting said second ligated probes to determine at least one nucleotide of said first target domain, wherein at least one nucleotide of said first target domain is determined by both said detecting of said first ligated probes and said detecting of said second ligated probes.
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Specification