Multiplex targeted amplification using flap nuclease

• US 9,523,122 B2
• Filed: 02/16/2015
• Issued: 12/20/2016
• Est. Priority Date: 01/17/2007
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Zheng, Jianbiao, Weng, Li, Faham, Malek
• Primary Examiner(s): THOMAS, DAVID C
• Application Number:
  US14/623,010
• Publication Number:
  US 20150152482A1
• Time in Patent Office: 673 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2531/125 Rolling circle

Abstract

Methods for multiplex amplification of a plurality of targets of distinct sequence from a complex mixture are disclosed. In one aspect targets are circularized using a single circularization probe that is complementary to two regions in the target that flank a region to be amplified. The targets may hybridize to the circularization probe so that 5′ or 3′ flaps are generated and methods for removing flaps and circularizing the resulting product are disclosed. In another aspect targets are hybridized to dU probes so that 5′ and 3′ flaps are generated. The flaps are cleaved using 5′ or 3′ flap endonucleases or 3′ to 5′ exonucleases. The target sequences are then ligated to common primers, the dU probes digested and the ligated targets amplified.

Claims

1. A method for amplifying a plurality of target sequences from a complex mixture of nucleic acid comprising:

a. fragmenting the nucleic acid to obtain a fragmented nucleic acid sample;

b. mixing the fragmented nucleic acid sample with a plurality of dU probes to the fragmented nucleic acid sample, wherein there is a dU probe for each target sequence in the plurality and wherein each dU probe comprises;

i. a central target region that is perfectly complementary to a target region in a target fragment in the nucleic acid sample, wherein the target fragment comprises a target sequence and a 5′

non-target sequence and wherein the target fragment hybridizes to the dU probe so that the target sequence is hybridized to the dU probe and the 5′

non-target sequences forms a single stranded 5′

flap structure;

ii. a 5′

first common sequence; and

iii. a 3′

second common sequence;

c. mixing the fragmented nucleic acid sample with a first oligonucleotide that is complementary to said first common sequence and a second oligonucleotide that is complementary to said second common sequence;

d. adding a 5′

flap nuclease to the mixture from (c) to cleave the 5′

flap from the target fragment to generate a new 5′

end in the target;

e. adding a ligase to the reaction to ligate the first oligonucleotide to the new 5′

end in the target and the second oligonucleotide to the 3′

end of the target;

f. cleaving the dU probes; and

g. amplifying the target sequences using primers for said first and second common sequences.

2. The method of claim 1 wherein the target sequence comprises a shared sequence of 2 to 8 bases and wherein said second oligonucleotide also comprises said shared sequence.

3. The method of claim 1 further comprising adding a 3′

to 5′

exonuclease to said mixture and wherein said exonuclease is selected from the group consisting of exonuclease I, exonuclease VII, exonuclease III and T7 exonuclease.

4. The method of claim 1 further comprising adding a 3′

flap endonuclease.

5. The method of claim 1 wherein the 5′

flap endonuclease is selected from the group consisting of FEN1 nuclease and Taq DNA polymerase I.

6. A method for amplifying a plurality of at least 100 target sequences from a nucleic acid sample comprising:

a. fragmenting the nucleic acid sample by a method that generates fragments that terminate at the 5′

end of one strand with a known sequence;

b. adding a plurality of dU probes to the fragments from (a), wherein each dU probe is specific for a target in the plurality of target sequences and wherein each dU probe comprises

i. a first 5′

region that is complementary to a first sequence in the target wherein said first sequence comprises at least 15 bases of the known sequence at the 5′

end of the target and includes the 5′

end of the target and

ii. a second 3′

region that is complementary to a second sequence in the target that is 3′

of the first sequence, is at least 15 bases and is separated from the first sequence by a third sequence,

wherein said dU probe hybridizes to said target to form a structure wherein the first sequence and the second sequence are brought into juxtaposition by hybridization of the dU probe and a 3′

single stranded flap sequence comprising the 3′

end of the target strand is generated;

c. adding a ligase, a 3′

flap nuclease, a DNA polymerase and at least one species of dNTP to form ligated targets;

d. digesting the dU probes; and

e. amplifying the ligated targets.

7. The method of claim 6 wherein said amplification is by rolling circle amplification, wherein a Phi29 DNA polymerase is used and the primer is selected from random primers, and target specific primers.

8. A method for amplifying a plurality of target sequences from a nucleic acid sample comprising:

a. fragmenting the nucleic acid sample by a method that generates fragments of known sequence at the 3′

end of the target strands to be amplified;

b. adding a plurality of dU probes to the fragments, wherein the plurality comprises a different dU probe for each target sequence to be amplified and wherein each dU probe comprises;

i. a first 5′

region that is complementary to a first sequence in the target;

ii. a second 3′

region that is complementary to a second sequence in the target,

wherein said second sequence is 3′

of said first sequence in the target strand to be amplified and hybridizes to the known sequence at the 3′

end of the target strand to be amplified and either includes the base at the 3′

end of the target strand or hybridizes so that the base at the 3′

end of the target forms a flap of a single base;

wherein said dU probe hybridized to the target strand to form a structure wherein the first sequence and the second sequence are brought into juxtaposition by hybridization of the dU probe and a 5′

single stranded flap sequence comprising the 5′

end of the target strand is generated;

c. adding a 5′

flap nuclease and a ligase to form ligated targets;

d. digesting the dU probes; and

e. amplifying the ligated targets.

9. The method of claim 8 wherein the structure comprises a 3′

flap of a single base and a 5′

flap of two or more bases and wherein the 5′

flap endonuclease is selected from the group consisting of FEN1 and Taq DNA Polymerase I.