Clonal pre-amplification in emulsion
First Claim
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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,the first adaptor molecule comprising a 5′
terminal part comprising a primer binding site, anda 3′
terminal part comprising an oligo (dT) sequence at least 5 nucleotides in length, a randomized sequence at least 5 nucleotides in length, or a specific sequence for a gene or gene family,(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.
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Abstract
Disclosed is a process for clonal pre-amplification of a nucleic acid involving the steps of (i) providing a plurality of different nucleic acid molecules (b) attaching adaptor sequences to the 3′ ends and 5′ ends of the nucleic acid molecules (c) preparing a water in oil emulsion wherein the majority of water droplets comprises one or none member of the plurality of different nucleic acid molecules (d) clonally amplifying the plurality of different nucleic acid molecules. In particular, the different nucleic acid molecules are mRNA molecules.
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Citations
18 Claims
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1. A method of generating an unbiased population of amplified cDNA comprising the steps of:
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(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,the first adaptor molecule comprising a 5′
terminal part comprising a primer binding site, anda 3′
terminal part comprising an oligo (dT) sequence at least 5 nucleotides in length, a randomized sequence at least 5 nucleotides in length, or a specific sequence for a gene or gene family,(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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Specification