Global amplification using a randomly primed composite primer
View Patent ↗The invention relates to the field of polynucleotide amplification. More particularly, the invention provides methods, compositions and kits for amplification of (i.e., making multiple copies of) a multiplicity of different polynucleotide template sequences using a randomly primed RNA/DNA composite primer.
1. A method for amplification of a template DNA, comprising:
(a) incubating the template DNA with
(i) a pool of first composite primers, each first composite primer comprising
(1) a 5′ RNA portion that is not hybridizable to the template DNA and
(2) a 3′ DNA portion, wherein the 3′ DNA portion of each of the first composite primers comprises a random sequence that is different from the 3′ DNA portion of the other first composite primers in the pool, and
(ii) a DNA polymerase having strand displacement activity,
whereby a complex comprising an RNA/DNA heteroduplex is generated;
(b) incubating the complex with an amplification primer, wherein the amplification primer is a composite primer comprising a 5′ RNA portion and a 3′ DNA portion, whereby multiple copies of an amplification product are generated, wherein the incubation of the complex with the amplification primer is under conditions that permit RNA cleavage, primer hybridization, primer extension, and displacement of the primer extension product when the RNA of the amplification primer is cleaved and another amplification primer binds to the complex and is extended by strand displacement, whereby multiple copies of a polynucleotide amplification product are generated.
2. The method of claim 1 , wherein the amplification primer comprises some of the sequence of the first composite primers in the pool.
3. The method of claim 1 , wherein each of the first composite primers in the pool and the amplification primer are different primers.
4. The method of claim 1 , step (a), wherein the template DNA is further incubated with an auxiliary primer comprising DNA, wherein the auxiliary primer is designed to randomly hybridize to a multiplicity of sites on the template DNA.
5. The method of claim 1 , wherein the DNA polymerase comprises a DNA-dependent DNA polymerase.
6. The method of claim 1 , wherein the DNA polymerase comprises an RNA-dependent DNA polymerase.
7. The method of claim 1 , wherein the incubation in step (a) is under conditions that permit hybridization of the pool of first composite primers to the template DNA and first primer extension, whereby the complex comprising an RNA/DNA heteroduplex is generated.
8. The method of claim 1 , wherein the amplification products are generated using a DNA-dependent DNA polymerase and an agent that cleaves RNA from an RNA/DNA heteroduplex.
9. The method of claim 6 , wherein the DNA polymerase comprises a DNA-dependent DNA polymerase and the RNA-dependent DNA polymerase in the same enzyme.
10. The method of claim 8 , wherein the agent is an enzyme.
11. The method of claim 10 , wherein the enzyme is RNase H.
12. The method of claim 1 , wherein the 5′ RNA portion of the amplification primer is adjacent to the 3′ DNA portion.
13. The method of claim 1 , wherein the 5′ RNA portion of each of the first composite primers consists of about 7 to about 50 nucleotides.
14. The method of claim 13 , wherein the 3′ DNA portion of each of the first composite primers consists of about 5 to about 20 nucleotides.
15. The method of claim 1 , wherein the 5′ RNA portion of the amplification primer consists of about 7 to about 50 nucleotides.
16. The method of claim 15 , wherein the 3′ DNA portion of the amplification primer consists of about 5 to about 20 nucleotides.
17. The method of claim 1 , wherein the incubation of step (b) further comprises a non-canonical nucleotide.
18. The method of claim 17 , wherein the non-canonical nucleotide is dUTP.
19. The method of claim 1 , wherein the incubation of step (b) further comprises a labeled nucleotide.
20. The method of claim 1 , wherein the amplification comprises global amplification.
21. The method of claim 20 , wherein the global amplification comprises amplification of genomic DNA or cDNA.
22. The method of claim 20 , wherein the global amplification comprises amplification of mitochondrial DNA, microbial DNA, viral DNA, eukaryotic DNA or mixtures thereof.
23. The method of claim 1 , wherein the pool of first composite primers comprises at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, or at least 100 different sequences.
24. The method of claim 1 , wherein each of the first composite primers is hybridizable to a multiplicity of template polynucleotide sites.
25. The method of claim 1 , wherein at least 50, at least 100, at least 200, at least 300, or more distinct sequences are amplified.
26. The method of claim 1 , wherein the template DNA comprises target polynucleotides, wherein the target polynucleotide sequences are unknown.
27. The method of claim 1 , wherein the template sequence is unknown.