Locus specific amplification using array probes

Patent metadata

Field	Value
Publication number	US-11408094-B2
Application number	US-201916402539-A
Country	US
Kind code	B2
Filing date	May 3, 2019
Priority date	Sep 14, 2007
Publication date	Aug 9, 2022
Grant date	Aug 9, 2022

Abstract

Methods are provided for multiplexed amplification of selected targets and analysis of the amplified targets. In preferred aspects the amplification and analysis take place on the same solid support and preferably in a localized area such as a bead or a feature of an array. In preferred aspects the analysis is a determination of sequence at one or more locations in the amplified target. The methods may be used for genotyping, sequencing and analysis of copy number.

First claim

What is claimed is:

1. A method for amplifying a plurality of targets said method comprising:
(a) hybridizing each target to a first complementary support bound probe in an array comprising a plurality of support bound probes, wherein probes of the same sequence are present in the same feature and wherein different targets hybridize to different first probes in different features;
(b) extending the first probes using the hybridized targets as template to obtain extended first probes;
(c) optionally removing the target;
(d) ligating a hairpin oligonucleotide to the 3′ end of the extended first probes, wherein said hairpin oligonucleotide comprises a double stranded region, a loop region and a 3′ end, and extending the 3′ end of the hairpin oligonucleotide using the extended first probes as template to obtain a double stranded support bound extension product corresponding to a double stranded copy of the target;
(e) allowing the 3′ end of the extension product generated in (d) to hybridize to second probes in the same features and extending the second probes to obtain second extended probes;
(f) allowing the 3′ end of the second extended probes to hybridize to another copy of the second probes and extending; and
(g) repeating step (f) at least once to obtain a plurality of amplified targets.

2. The method of claim 1 , wherein the hairpin oligonucleotide includes a cleavage site.

3. The method of claim 1 , wherein the double stranded region of the hairpin terminates with a base pair between the 5′ terminal nucleotide and the 3′ terminal nucleotide.

4. The method of claim 2 , wherein the cleavage site is one or more uracil bases and cleavage is by treatment with a uracil DNA glycosylase to generate an abasic site and cleavage of the abasic site with an abasic endonuclease.

5. The method of claim 4 , wherein the abasic site is cleaved by E. coli Endonuclease V.

6. The method of claim 4 , wherein the abasic site is cleaved by Tma Endonuclease V.

7. The method of claim 2 , wherein the cleavage site is a restriction site.

8. The method of claim 1 , wherein the hairpin oligonucleotide includes a primer binding site.

9. The method of claim 1 , wherein the end of the hairpin oligonucleotide is blunt so that the base at the 3′ end of the hairpin oligonucleotide is complementary to the base at the 5′ end of the hairpin oligonucleotide.

10. The method of claim 1 , wherein the 3′ end of the hairpin oligonucleotide is recessed to the 5′ end of the hairpin oligonucleotide.

11. The method of claim 1 , wherein the 3′ end of the hairpin oligonucleotide is extended one or more bases beyond the 5′ end of the hairpin oligonucleotide.

12. The method of claim 1 , wherein the 3′ end of the hairpin oligonucleotide has one or more degenerate bases.

13. A method for analyzing a plurality of targets comprising:
(a) amplifying the plurality of targets according to the method of claim 1 ;
(b) hybridizing a primer to the amplified targets;
(c) extending the hybridized primer by a single base using a template dependent polymerase, wherein the base that is added is complementary to the base in the target that is immediately adjacent to the 3′ end of the primer;
(d) determining the identity of the base added in (c); and
(e) repeating (c) and (d) to determine the sequence of a region of the target.

14. The method of claim 13 , wherein the reaction includes each of the bases A, G, C and T and they are differentially labeled so that each base carries a different label and wherein the bases are blocked from extension by a blocking group.

15. The method of claim 14 , wherein after extending by a single base that is blocked and labeled, the blocking group is removed and the label is removed and the extension is repeated for at least one base, thereby determining the sequence of a plurality of bases in the target.

Assignees

• Affymetrix Inc

Inventors

• Fu Glenn
• Shapero Michael
• Wang Pei-Hua

Classifications

• C12Q1/6837 Primary

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C12Q1/6858

  Allele-specific amplification · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C40B50/06 Primary

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title

• C12Q2531/113

  PCR · CPC title