Locus Specific Amplification Using Array Probes

• US 20170051339A1
• Filed: 06/16/2016
• Published: 02/23/2017
• Est. Priority Date: 09/14/2007
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Fu, Glenn K., Shapero, Michael H., Wang, Pei-Hua
• Granted Patent: US 10,329,600 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

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.

Claims

1. A method for amplifying a plurality of target sequences from a nucleic acid sample and analyzing the amplified target sequences, said method comprising:

(a) fragmenting the nucleic acid sample to obtain fragments;

(b) mixing the fragments obtained in (a) with an array of probes arranged in features, wherein a feature comprises multiple copies of a target specific probe, so that fragments hybridize to complementary target specific probes to form probe;

target complexes;

(c) extending the probes in the probe;

target complexes using a polymerase, wherein the target is used as template for extension of the probe and wherein an extended probe is generated;

(d) removing the target from the extended probe by denaturation;

(e) generating a plurality of complementary copies of the extended probe, wherein said copies are not covalently attached to the surface of the solid support;

(f) allowing the copies to hybridize to the complementary target specific probes in the same feature; and

(g) analyzing the copies.

2. The method of claim 1, wherein prior to step (f) amplified target sequences are fragmented to obtain amplified target fragments and the amplified target fragments are labeled with a detectable label to obtain labeled fragments and wherein said step of analyzing comprises hybridizing the labeled fragments to said array.

3. The method of claim 1 wherein said plurality of target sequences comprises between 100 and 100,000 different target sequences.

4. The method of claim 1 wherein said plurality of target sequences comprises between 1000 and 100,000 different target sequences.

5. The method of claim 1 wherein said plurality of target sequences comprises between 100,000 and 3,000,000 different target sequences.

6. The method of claim 1 wherein said step of analyzing is to determine the genotype of polymorphisms in said target sequences in said nucleic acid sample.

7. The method of claim 1 wherein said step of analyzing is to determine the methylation status of one or more cytosines in said target sequences in said nucleic acid sample.

8. The method of claim 1 wherein said step of analyzing is to determine the presence or absence of specific target sequences in said nucleic acid sample.

9. A method for amplifying and analyzing a plurality of target sequences from a nucleic acid sample, said method comprising:

(a) fragmenting the nucleic acid sample with a restriction enzyme to obtain fragments with known sequences at the 5′ and

3′

ends of the fragments, wherein said fragments comprise a plurality of target fragments comprising target sequences;

(b) mixing the fragments obtained in (a) with a plurality of target specific splint oligonucleotides, wherein each splint oligonucleotide comprises a first target complementary sequence that is at least 10 bases in length and is perfectly complementary to the at least 10 bases at the 3′

end of a corresponding target fragment, and a second target complementary sequence that is at least 10 bases in length and is perfectly complementary to the at least 10 bases at and including the 5′

end of said corresponding target fragment, and wherein said first sequence is 5′

of said second sequence in said splint oligonucleotide, wherein target specific splint oligonucleotides further comprises a first common priming sequence at the 5′

end and a second common priming sequence at the 3′

end;

(c) adding first and second primers to the mixture wherein said first primer is complementary to said first common priming sequence and said second primer is complementary to said second common priming sequence and wherein said first and second primers hybridize to said splint oligonucleotides so the first primer is adjacent to the 3′

end of the target fragment and the second primer is adjacent to the 5′

end of the target fragment;

(d) ligating the first primer to the 3′

end of the target fragment and the second primer to the 5′

end of the target fragment to obtain ligated target fragments comprising a first common priming site at the 3′

end and a second common priming sites at the 5′

end;

(e) after said ligating step (d) fragmenting said splint oligonucleotides;

(f) amplifying the ligated target fragments from (d) to obtain amplified target fragments; and

(g) analyzing the amplified target fragments by a method comprising hybridization to an array comprising a plurality of oligonucleotide probes present at known or determinable locations in the array.

10. The method of claim 9 wherein each splint oligonucleotide further comprises a target fragment specific tag sequences located between the first target complementary sequence and the first common priming sequence or between the second target complementary sequence and the second common priming sequence and wherein tag complement oligonucleotides that are complementary to the tag sequences in the splint oligonucleotides are added at step (c) and ligated to the target fragments in step (d) so that the tag complements are adjacent to one of the common priming sequence in the ligated target fragments and wherein the array is a tag array.

11. The method of claim 9 wherein the second primer comprises a nicking position and wherein said amplifying comprises:

(a) making the ligated target fragments double stranded;

(b) nicking the nicking position;

(c) extending from the nick using a strand displacing DNA polymerase; and

(d) repeating steps (b) and (c).

12. The method of claim 11 wherein said nicking is by cleavage with a nicking restriction enzyme.

13. The method of claim 11 wherein said nicking is by generating a nick 3′

of an inosine.

14. The method of claim 13, wherein said nick occurs about 2-3 nucleotides downstream of said 3′

end of said inosine base.

15. The method according to claim 11, wherein said second primer is between 15 and 100 bases in length.

16. The method according to claim 11, wherein said second primer is between 15 and 50 bases in length.

17. The method according to claim 11, wherein said second primer is between 15 and 30 bases in length.

18. The method of claim 11, wherein said DNA polymerase with strand displacement function is selected from the group consisting of Klenow fragment, Bst polymerase, and phi29 polymerase.

19. The method of claim 11, wherein said DNA polymerase is active at a temperature between 30°

C. and 80°

C.

20. The method according to claim 11, wherein the DNA polymerase is Bst DNA polymerase and is active between 50°

C. to 65°

C.

21. The method according to claim 11, wherein the DNA polymerase is phi29 DNA polymerase and is active at between 30°

C. and 37°

C.

22. The method according to claim 13, wherein said nicking is by Endo V.

23. The method of claim 22, wherein said Endo V is from E. coli.

24. The method of claim 22, wherein said Endo V is a thermal stable version.

25. The method according to claim 22, wherein said Endo V is active between 30°

C. and 60°

C.

26. The method according to claim 22, wherein said Endo V is active at a temperature between 30°

C. and 37°

C.

27. The method of claim 9 wherein the splint oligonucleotides comprise one or more uracil bases and wherein the splint oligonucleotides are cleaved by uracil DNA glycosidase treatment, wherein the uracil is converted to an abasic site by uracil DNA glycosidase and the abasic sites are cleaved.

28. The method of claim 22 where the abasic sites are cleaved by E. coli Endonuclease V and the strand displacing polymerase is phi29.

29. The method of claim 27 where the abasic sites are cleaved by Tma Endonuclease V and the strand displacing polymerase is Bst DNA polymerase.