Multiplex amplification methods
View Patent ↗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.
1. A method for amplifying a plurality of target nucleic acids comprising:
(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 probes 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 comprise 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 of the extended capture probes with a single strand specific 3′ endonuclease;
(f) forming a duplex between the 5′ overhang region of the splint probe and a ligatable sequence comprising a region that is complementary to a region of the splint probe and 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 3′ end, a template complementary region, the first priming sequence, wherein the first priming sequence is common to all capture probes, a cleavage site, the second priming sequence, wherein the second priming sequence is common to all capture probes, the tag sequence, and a region that is complementary to the overhang region of the splint probe, wherein step (f) comprises:
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, 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 comprise in the 5′ to 3′ direction, a first target complementary region, the first priming sequence, wherein the first priming sequence is common to all capture probes, the tag sequence and a second target complementary region.
6. The method of claim 1 wherein the splint probe comprises the target complementary region, the 5′ overhang region, and thee ligatable sequence comprising a region that is complementary to another region of the splint probe and thee second priming sequence, and wherein step (f) comprises:
forming an intra-molecular loop between the 5′ overhang region and the ligatable sequence of the splint probe, 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, the method further comprising 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, the method further comprising 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.