Patent ID: 9650629
Date: 2017-05-16
CPC Classifications: C12N

Claim:
1. A method of generating an unbiased population of amplified cDNA comprising the steps of: (a) providing a plurality of different single-stranded RNA molecules, the plurality comprising different single-stranded RNA molecules present at different representation frequencies in the plurality, (b)-(b1) hybridizing a first adaptor molecule to the 3′ ends of RNA molecules of said plurality, (b2) performing a first strand cDNA synthesis in the presence of an RNA-dependent DNA polymerase and a dNTP mixture to generate a pool of single-stranded cDNA molecules, (b3) attaching a second adaptor molecule to cDNA molecules of said pool, (c) preparing a water-in-oil emulsion by statistically distributing the cDNA molecules obtained in step (b3) into an excess of aqueous droplets of a water-in-oil emulsion so that a majority of water droplets comprise one or zero cDNA molecules; and (d) clonally amplifying the cDNA molecules within the emulsion using polymerase chain reaction to produce an amplified population comprising different cDNA molecules present at representation frequencies that are about equal to the representation frequency of the different single-stranded RNA molecules present in the plurality.