Nucleic acid analysis using sequence tokens
First Claim
1. A method of screening for the presence or absence of a nucleotide allele of a polymorphism in multiple DNA samples, the method comprising the steps of:
- combining the following under hybridization conditions;
(i) a mixture comprising single stranded DNA fragments derived from multiple DNA samples, wherein the single stranded DNA fragments comprise;
(a) a unique tag corresponding to the DNA sample from which it is derived; and
(b) a sequence of interest having a polymorphic site, the polymorphic site having a wild type nucleotide allele and a second nucleotide allele; and
(ii) RNA probes comprising a sequence complementary to the sequence of interest having the wild type nucleotide allele at the polymorphic site;
thereby forming RNA/DNA heteroduplexes;
removing any mismatched nucleotide at the polymorphic site from the RNA probes in the heteroduplexes;
contacting the heteroduplexes with a nucleotide triphosphate complementary to the second nucleotide allele under nucleic acid polymerization conditions;
isolating heteroduplexes that have incorporated the nucleotide triphosphate complementary to the second nucleotide allele; and
determining the unique tags of the single stranded DNA fragments of the isolated heteroduplexes to identify genomic DNA samples having the second nucleotide allele at the polymorphic site in the sequence of interest, thereby screening the multiple DNA samples for the presence or absence of the second nucleotide allele.
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Abstract
The present invention provides methods and compositions for tagging nucleic acid sequence fragments, e.g., a set of nucleic acid sequence fragments from a single genome, with one or more unique members of a collection of oligonucleotide tags, or sequence tokens, which, in turn, can be identified using a variety of readout platforms. As a general rule, a given sequence token is used once and only once in any tag sequence. In addition, the present invention also provides methods for using the sequence tokens to efficiently determine variations in nucleotide sequences in the associated nucleic acid sequence fragments.
101 Citations
20 Claims
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1. A method of screening for the presence or absence of a nucleotide allele of a polymorphism in multiple DNA samples, the method comprising the steps of:
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combining the following under hybridization conditions; (i) a mixture comprising single stranded DNA fragments derived from multiple DNA samples, wherein the single stranded DNA fragments comprise; (a) a unique tag corresponding to the DNA sample from which it is derived; and (b) a sequence of interest having a polymorphic site, the polymorphic site having a wild type nucleotide allele and a second nucleotide allele; and (ii) RNA probes comprising a sequence complementary to the sequence of interest having the wild type nucleotide allele at the polymorphic site; thereby forming RNA/DNA heteroduplexes; removing any mismatched nucleotide at the polymorphic site from the RNA probes in the heteroduplexes; contacting the heteroduplexes with a nucleotide triphosphate complementary to the second nucleotide allele under nucleic acid polymerization conditions; isolating heteroduplexes that have incorporated the nucleotide triphosphate complementary to the second nucleotide allele; and determining the unique tags of the single stranded DNA fragments of the isolated heteroduplexes to identify genomic DNA samples having the second nucleotide allele at the polymorphic site in the sequence of interest, thereby screening the multiple DNA samples for the presence or absence of the second nucleotide allele. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification