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 under conditions that permit cleavage of RNA in the RNA/DNA heteroduplex; and
(c) 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, wherein the amplification primer anneals to sequence complementary to the RNA sequence cleaved from the RNA/DNA heteroduplex, whereby multiple copies of an amplification product are generated.
2. The method of claim 1 , wherein the amplification primer comprises 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 , wherein the incubating in step (a) is under conditions that permit hybridization of the pool of first composite primers to the template DNA and first primer extension.
5. 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.
6. The method of claim 1 , wherein the DNA polymerase comprises a DNA-dependent DNA polymerase and an RNA-dependent DNA polymerase.
7. The method of claim 5 , wherein the agent is an enzyme.
8. The method of claim 7 , wherein the enzyme is RNase H.
9. The method of claim 1 , wherein the 5′ RNA portion of the amplification primer is adjacent to the 3′ DNA portion of the amplification primer.
10. The method of claim 1 , wherein amplification comprises global amplification.
11. The method of claim 10 , wherein the global amplification comprises amplification of genomic DNA or cDNA.
12. The method of claim 10 , wherein the global amplification comprises amplification of mitochondrial DNA, microbial DNA, viral DNA, eukaryotic DNA or mixtures thereof.
13. 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.
14. The method of claim 1 , wherein each of the first composite primers is hybridizable to a multiplicity of template polynucleotide sites.
15. The method of claim 1 , wherein at least 50, at least 100, at least 200, at least 300, or more distinct sequences are amplified.
16. The method of claim 1 , wherein the template DNA comprises target polynucleotides, wherein sequences of the target polynucleotide sequences are unknown.
17. The method of claim 1 , wherein the template DNA is comprises unknown sequence.