Kits for multiplexed nucleic acid analysis by capture of single-stranded DNA produced from double-stranded target fragments
View Patent ↗A method of fragmentation of double stranded DNA is disclosed for use in nucleic acid analysis, notably in the multiplexed analysis of polymorphisms and mutations. The method produces a multiplicity of labeled sense and anti-sense fragments which are not complementary, and thus do not significantly re-anneal under conditions suitable for hybridization analysis (or capture-mediated elongation analysis) of the polymorphisms and/or mutations. The fragments display a desired or predicted length distribution. Cleavage sites can be selected such that the fragments are short, yet long enough to allow discrimination among fragments in an assay, and as a matter of statistical probability, such that the majority of fragments contain at least one labeled nucleotide to facilitate detection.
1. A method for detecting particular nucleotide sequences in a double-stranded oligonucleotide sample, comprising:
amplifying segments of the sample with unlabeled primers in the presence of labeled nucleotides to generate labeled double stranded amplicons;
cleaving the double-stranded amplicons at a multiplicity of sites which are randomly distributed along each strand to produce fragments which are not fully complementary to one another;
mixing the fragments with a hybridization buffer;
placing the fragments in contact with a set of single-stranded oligonucleotides under annealing conditions, wherein single-stranded oligonucleotides in the set are complementary to the fragments or to subsequences of the fragments; and
detecting hybridization between said set of single-stranded oligonucleotides and the fragments.
2. The method according to claim 1 , wherein the labeled nucleotides are fluorescently labeled.
3. The method according to claim 1 , wherein the labeled nucleotides comprise biotin.
4. The method of claim 3 , wherein detecting comprises contacting the fragments with labeled streptavidin.
5. The method according to claim 1 , wherein cleaving comprises depurination of the amplicon by contacting the amplicon with hydrochloric acid then heat-denaturing to generate single-stranded DNA fragments.
6. The method of claim 1 , wherein fragment length is controlled by controlling the pH of the hydrochloric acid, time of exposure to the hydrochloric acid and reaction temperature.
7. The method of claim 6 , wherein fragment length is adjusted so as to generate sense and/or anti-sense fragments comparable in length to the single-stranded oligonucleotides.
8. The method of claim 1 , wherein the single-stranded oligonucleotides are DNA.
9. The method of claim 1 , wherein the double-stranded oligonucleotide sample is genomic DNA.
10. The method of claim 1 , wherein the double-stranded oligonucleotide sample includes genetic loci of interest.