Compositions for amplification of RNA sequences using composite primers
View Patent ↗The invention provides methods for isothermal amplification of RNA. The methods are particularly suitable for amplifying a plurality of RNA species in a sample. The methods employ a composite primer, a second primer and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In another aspect, the methods employ a single primer (which is a composite primer) and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In some embodiments, a transcription step is included to generate multiple copies of sense RNA of an RNA sequence of interest. The methods are useful for preparation of nucleic acid libraries and substrates for analysis of gene expression of cells in biological samples. The invention also provides compositions and kits for practicing the amplification methods, as well as methods which use the amplification products.
1. A composition comprising at least one RNA template and a plurality of primers, wherein the primers are composite primers that comprise a 5′ RNA portion and a 3′ DNA portion, wherein the 3′ DNA portion comprises a random sequence, and wherein the random sequence in at least one of the plurality of primers is complementary to a sequence in the at least one RNA template.
2. The composition of claim 1 , further comprising an RNA-dependent DNA polymerase.
3. The composition of claim 1 , further comprising a DNA-dependent DNA polymerase.
4. The composition of claim 1 , further comprising an enzyme that cleaves RNA from an RNA/DNA hybrid.
5. The composition of 4 , wherein the enzyme is RNaseH.
6. The composition of claim 1 , further comprising a labeled dNTP.
7. The composition of claim 6 , wherein the labeled dNTP consists of a label selected from a group consisting of fluorescent dyes, radioisotopes, enzymes, steroids, colorimetric labels, ligands, and anti-ligands.
8. The composition of claim 1 , wherein the RNA portion of the primers consists of about 10 to about 20 nucleotides and the DNA portion of the primers consists of about 5 to about 20 nucleotides.
9. The composition of claim 1 , wherein the 5′ RNA portion of the primers is adjacent to the 3′ DNA portion.
10. The composition of claim 1 , wherein the primers are tailed primers that comprise a 5′ portion that is not hybridizable to the at least one RNA template under conditions in which the primers hybridize to the at least one RNA template.
11. The composition of claim 1 , wherein the random sequence of the 3′DNA portion of the primers comprises at least 1 random nucleotide at the 3′ end.
12. The composition of claim 1 , wherein the at least one RNA template is selected from a group consisting of total RNA, tRNA, mRNA, rRNA, mitochondrial RNA, chloroplast RNA, DNA-RNA hybrids, viral RNA, cell free RNA, and mixtures thereof.
13. The composition of claim 1 , wherein the 5′ RNA portion comprises a defined sequence.
14. The composition of claim 1 , wherein the plurality of primers comprises a first pool of composite primers, and a second pool of composite primers, wherein the first pool of composite primers comprises a mixture of composite primers which comprise the same 5′ RNA portion and a multiplicity of random sequences in the 3′ DNA portion, and wherein the second pool of composite primers comprises a mixture of composite primers which comprise the same 5′ RNA portion and a multiplicity of random sequences in the 3′ DNA portion, wherein the 5′ RNA portion and 3′ DNA portion of the first pool of composite primers is different from the second pool of composite primers.
15. The composition of claim 1 , further comprising an additional composite primer comprising a 5′ RNA portion and a 3′ DNA portion, wherein the 3′ DNA portion of the composite primer comprises a poly-dT sequence.