Multiplex Amplification Methods

• US 20110294689A1
• Filed: 05/27/2011
• Published: 12/01/2011
• Est. Priority Date: 05/27/2010
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Namsaraev, Eugeni A.
• Granted Patent: US 8,828,688 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• 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 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• 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 2549/113 using nested probes
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

Compositions and methods for amplifying selected polynucleotides, including DNA and RNA, particularly in multiplex amplification reactions using common primers amplification. Generally, methods of the invention employ multiple steps such as template-specific hybridization, a linear amplification, partial degradation of nucleic acid, and ligation. At the end of the process the sequences of selected polynucleotides are flanked by the common sequences which can be used for exponential amplification using common primers. In some aspects the polynucleotides are associated with a barcode and the presence of the barcode is detected to measure the amount of the polynucleotide.

Claims

1. A method for amplifying a plurality of target nucleic acids comprising obtaining a plurality of capture probes comprising a capture probe for each target nucleic acid in the plurality of target n:

(a) obtaining a plurality of capture probes comprising a capture probe for each target nucleic acid in the plurality of target nucleic acids wherein each capture probe comprises a first 3′

region that is complementary to a target nucleic acid in the plurality of target nucleic acids, a second region that has a first priming sequence and optionally a third region between said first and second regions that comprises a tag sequence;

(b) mixing the capture probe with a nucleic acid sample that includes the target nucleic acids under conditions that allow hybridization of the capture probes to form duplexes between the capture probes and the target nucleic acids;

(c) extending the capture probes from the 3′

end with a polymerase using the target nucleic acids as template to form extended capture probes that comprises a newly added region that is complementary to the target nucleic acid;

(d) hybridizing a splint probe, comprising a target complementary region and a 5′

overhang region, to a portion of the newly added region to form a duplex between the target complementary region of the splint probe and a portion of the newly added region leaving a 3′

region of the extended capture probe single stranded, wherein the 3′

region includes the 3′

end of the extended capture probe;

(e) degrading the 3′

region with a single strand specific 3′

endonucleases;

(f) forming a duplex between the overhang region of the splint probe and a ligatable sequence comprising a second priming sequence and ligating said ligatable sequence to the 3′

end of the extended capture probe to form a ligated product; and

(g) amplifying the ligated product using the first and second priming regions.

2. The method of claim 1 wherein said ligatable sequence is an oligonucleotide having a 5′

region that is complementary to a region of the splint probe and a 3′

region that comprises the second priming sequence.

3. The method of claim 1 wherein the capture probe comprises from the 5′

end, a template complementary region, a first common sequence, a cleavage site, a second common sequence, a barcode region and a region that is complementary to the overhang region of the splint probe, the method further comprising;

forming a duplex between the overhang region of the splint probe and the region that is complementary to the overhang region of the splint probe and thereby bringing the 5′

end of the extended capture probe so that it is adjacent to the 3′

end of the extended capture probe; and

ligating the 5′

end of the extended capture probe to the 3′

end of the extended capture probe thereby forming a closed circle prior to step (g).

4. The method of claim 3 further comprising cleaving the closed circle at the cleavage site in the capture probe prior to step (g).

5. The method of claim 2 wherein the capture probes comprises in the 5′

to 3′

direction, a first target complementary region, a first common sequence, a barcode sequence and a second target complementary region.

6. The method of claim 1 wherein the capture probes comprising a first 3′

region that is complementary to the target nucleic acid, a second region that has a first priming sequence and optionally a third region between said first and second regions that comprises a tag sequence; and

wherein the method further comprises;

forming an intra-molecular loop between the overhang region so that the 5′

end of the splint probe is adjacent to the 3′

end of the extended capture probe; and

ligating the 5′

end of the splint probe to the 3′

end of the extended capture probe prior to step (g).

7. The method of claim 1 wherein the capture probe comprises a plurality of uracil residues and further comprising the degrading the extended capture probe after making at least one complimentary copy.

8. The method of claim 1 wherein the splint probe comprises a plurality of uracil residues and further comprising the degrading the extended capture probe after making at least one complimentary copy.

9. The method of claim 7 wherein the splint probe comprises a plurality of uracil residues and further comprising the degrading the extended capture probe after making at least one complimentary copy.

10. A method for amplifying an RNA target comprising:

(a) obtaining a capture probe comprising from the 5′

end a second priming sequence, a tag sequence, a first priming sequence and a sequence complementary to the RNA target;

(b) hybridizing the RNA target to the capture probe and extending the RNA using a polymerase and using the capture probe as template to form a duplex between the capture probe and the extended RNA target;

(c) optionally destroying or removing the capture probe; and

(d) amplifying the portion of the extended RNA comprising the first and second primer regions and the tag region.

11. The method of claim 10 wherein the capture probe further comprises an oligonucleotide complementary region between the first priming sequence and the sequence complementary to the RNA target, hybridizing a DNA oligonucleotide that is complementary to said oligonucleotide complementary region to the capture probe, ligating said oligonucleotide to the RNA target, wherein said extending step is extending from the end of the newly ligated DNA oligonucleotide.

12. The method of claim 10 wherein the capture probe comprises from the 5′

end a second priming sequence comprising a plurality of uracil bases, a tag sequence, a sequence complementary to the RNA target, and a first priming sequence;

treating the duplex with UDG and an AP endonuclease to remove the second priming sequence from the capture probe thereby generating a 3′

overhanging region in the extended RNA target and a new 5′

end in the capture probe; and

ligating the 3′

end of the extended RNA target to the new 5′

end of the capture probe to obtain a ligated product comprising the extended RNA target and a portion of the capture probe prior to step (d).

13. The method of claim 10 wherein the capture probe comprises from the 5′

end a tag sequence, a second priming sequence, a sequence complementary to the RNA target and a first priming sequence, wherein the tag sequence and the second priming sequence contain uracil bases;

the method further comprising;

cleaving the capture probe with UDG and AP endonucleases after step (b); and

ligating the 3′

end of the extended RNA target to the new 5′

end of the capture probe to obtain a ligated product comprising the extended RNA target and a portion of the capture probe prior to step (d).

14. The method of claim 10 wherein the capture probe comprises from the 5′

end;

a restriction enzyme recognition site, a tag sequence, a first priming sequence, and a sequence complementary to the RNA target and after extending the RNA using a polymerase using the capture probe as template the duplex comprises a double stranded restriction enzyme recognition site;

the method further comprising;

cleaving the capture probe at the restriction enzyme recognition site to generate a 3′

overhang and a recessed 5′

end;

ligating a double stranded adapter to the overhang wherein the double stranded adapter comprises a second priming sequence; and

wherein step (d) comprises amplifying the portion of the ligated product comprising the first and second primer regions and the tag region.

15. The method of claim 10 wherein the capture probes comprise from the 5′

end;

an oligonucleotide complementarity region, a tag sequence, a first priming sequence, a first priming region and a sequence complementary to the RNA target and after extending the RNA using a polymerase using the capture probe as template there duplex comprises the complement of the oligonucleotide complementarity region at the 3′

end of the extension product;

the method further comprising the following steps prior to step (d);

denaturing the capture probe extension product duplex and hybridizing a partially double stranded adapter to the extension product, wherein the bottom strand comprises a region complementary to the complement of the oligonucleotide complementarity region and a second priming region and the top strand is complementary to the second priming region; and

ligating the top strand to the 3′

end of the extension product;

wherein step (d) comprises amplifying the portion of the ligated product comprising the first and second primer regions and the tag region.

16. The method of claim 10 wherein the capture probes comprise from the 5′

end;

an oligonucleotide complementarity region, a tag sequence, a first priming sequence, a first priming region and a sequence complementary to the RNA target;

wherein the extended RNA target comprises the complement of the oligonucleotide complementarity region at the 3′

end of the extended RNA target;

the method further comprising denaturing the capture probe extension product duplex and hybridizing an oligonucleotide comprising a 5′

second priming region having a region of self complementarity and a 3′

region that is complementary to the 3′

end of the extension product; and

ligating the 5′

end of the oligonucleotide to the 3′

end of the extension product; and

wherein the step of amplifying comprises amplifying the portion of the ligated product comprising the first and second primer regions and the tag region.

17. The method of claim 10 wherein the capture probes comprise from the 5′

end;

an oligonucleotide complementary region, a tag sequence, a first priming sequence and a sequence complementary to the RNA target;

wherein the extended RNA target comprises the complement of the oligonucleotide complementarity region at the 3′

end of the extended RNA target;

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the method further comprising denaturing the capture probe extension product duplex and hybridizing an oligonucleotide comprising a 5′

second priming sequence and a 3′

region that is complementary to the 3′

end of the extension product; and

extending the 3′

end of the extension product to incorporate the complement of the second priming sequence; and

wherein the step of amplifying comprises amplifying the portion of the ligated product comprising the first and second primer regions and the tag region.

18. The method of claim 10 wherein the capture probes comprise from the 5′

end;

a second priming sequence, a tag sequence, a first priming sequence and a sequence complementary to an internal portion of the RNA target;

wherein the RNA target hybridizes to the capture probe leaving a 3′

overhanging portion of the RNA target and a 5′

overhanging portion of the RNA target; and

further comprising;

treating the duplex with RNase to remove the 5′

overhang and most of the 3′

overhang; and

removing the 3′

flap to generate a 3′

end that is base paired with the capture probe;

prior to extending the RNA.

19. The method of claim 10 wherein the capture probes comprise from the 5′

end;

a second priming sequence, a tag sequence, a first priming sequence and a sequence complementary to an internal portion of the RNA target;

wherein the RNA target hybridizes to the capture probe leaving a 3′

overhanging portion of the RNA target and a 5′

overhanging portion of the RNA target; and

further comprising;

treating the duplex with RNase H to generate a nick in the portion of the RNA that is hybridized to the DNA prior to extending the RNA.

20. A method for detecting a target nucleic acid in a sample comprising:

mixing the nucleic acid sample with a collection of encoded particles having target specific probes attached thereto,

wherein each different particle type has a first probe type and a second probe type attached, said first probe type having a region complementary to a first region of a target and said second probe having a region complementary to a second region of the same target,

wherein said first and second regions are distinct; and

wherein the duplex formed when both first and second probe types are hybridized to the target has a higher stability than the duplex between the target and either the first or second probe alone.

21. The method of claim 20 further comprising extending the first probe to fill the gap between the first and second probes on the target and ligating the ends of the first and second probe together to form a ligated probe.

22. The method of claim 21 further comprising cleaving the ligated probe to generate a new probe termini and labeling the new probe termini with a detectable label.