Patent ID: 7939258

Claim:
A method for amplification of a polynucleotide template, comprising: (a) extending a first primer hybridized to the polynucleotide template with at least one enzyme comprising a DNA polymerase activity, wherein the first primer is a single-stranded composite primer comprising an RNA portion and a 3′ DNA portion, whereby a first complex comprising a first primer extension product and the template polynucleotide is produced; (b) extending a second primer hybridized to the first primer extension product to produce a second complex comprising the first primer extension product and a second primer extension product wherein the second complex comprises a double-stranded RNA-DNA heteroduplex at one end; (c) incubating a reaction mixture comprising: (i) the second complex comprising the first primer extension product and the second primer extension product wherein the second complex comprises a double-stranded RNA-DNA heteroduplex at one end; (ii) unhybridized first primer: and (iii) an enzyme that is capable of cleaving single-stranded RNA, whereby the single-stranded RNA portion of excess unhybridized first primer is cleaved prior to amplification; (d) inactivating the enzyme that is capable of cleaving single-stranded RNA; (e) cleaving RNA from the double-stranded RNA-DNA heteroduplex with at least one enzyme that is capable of cleaving RNA from a double-stranded RNA-DNA heteroduplex; (f) introducing a composite amplification primer wherein the composite amplification primer hybridizes to the second primer extension product, and wherein the composite amplification primer comprises an RNA portion and a 3′ DNA portion; and (g) extending the composite amplification primer hybridized to the second primer extension product with at least one enzyme comprising DNA-dependent DNA polymerase activity, whereby the first primer extension product is displaced, RNA is cleaved from the composite amplification primer and another composite amplification primer hybridizes such that primer extension and strand displacement are repeated, and whereby multiple copies of a polynucleotide amplification product are generated.