Kits for multiplexed nucleic acid analysis by capture of single-stranded DNA produced from double-stranded target fragments

• US 8,563,247 B2
• Filed: 07/29/2010
• Issued: 10/22/2013
• Est. Priority Date: 10/29/2003
• Status: Active Grant

• Current Assignee: BioArray Solutions Limited (Werfen SA)
• Original Assignee: BioArray Solutions Limited (Werfen SA)
• Inventors: Yang, Jiacheng
• Primary Examiner(s): Martinell, James
• Application Number:
  US12/846,619
• Publication Number:
  US 20120015836A1
• Time in Patent Office: 1,181 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

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.

Claims

1. A method of detecting particular nucleotide sequences in a double-stranded oligonucleotide sample, comprising:

cleaving the double-stranded oligonucleotide at locations which are not aligned between the oligonucleotide strands, to thereby generate sense and anti-sense fragments which are not fully complementary;

placing the fragments 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 single-stranded oligonucleotides and the fragments.

2. The method of claim 1 wherein the single-stranded oligonucleotides are DNA.

3. The method of claim 1 wherein the double-stranded oligonucleotide-sample is genomic DNA.

4. The method of claim 1 wherein the double-stranded oligonucleotide sample includes genetic loci of interest.

5. The method of claim 1 wherein the cleaving is achieved by randomly reacting the purine bases on the strands with hydrochloric acid to depurinate them, and then heat-denaturing to generate single-stranded DNA fragments.

6. The method of claim 5 wherein the selection of cleavage sites is controlled by controlling the pH of the hydrochloric acid, the time of exposure to the hydrochloric acid and the reaction temperature.

7. The method of claim 1 wherein the fragments are labeled and the frequency of labeling is adjusted depending on the predicted fragment length, such that where shorter fragments are predicted following cleaving, the frequency of labeling is increased.

8. The method of claim 1 wherein the fragment length is adjusted so as to generate fragments approximating the length of the single-stranded oligonucleotides.

9. The method of claim 1 further including the step of providing conditions suitable for elongation of the single-stranded oligonucleotides, and detecting elongation of the single-stranded oligonucleotides.

10. The method of claim 9 wherein hybridization of particular single-stranded oligonucleotides and fragments is detected based on whether there is elongation of said particular single-stranded oligonucleotide.

11. The method of claim 1 further including the step of providing a set of single-stranded oligonucleotide probes some of which may be shorter than or complementary to the single-stranded oligonucleotides.

12. The method of claim 11 further including the step of providing conditions suitable for elongation of the single-stranded oligonucleotide probes, and detecting elongation of the single-stranded oligonucleotide probes.

13. The method of claim 12 further including the step of comparing the results from elongation of the single-stranded oligonucleotide probes with the results from the elongation of the single-stranded oligonucleotides.

14. The method of claim 13 wherein the comparison is performed automatically using a software program.