Multiplex Amplification Methods
First Claim
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.
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Accused Products
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.
82 Citations
22 Claims
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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:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method for amplifying an RNA target comprising:
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(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. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A method for detecting a target nucleic acid in a sample comprising:
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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. - View Dependent Claims (21, 22)
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Specification