Patent ID: 8617817
Filing Date: 2013-12-31
Classification: C12N,C12Q

Abstract:
1. A method for producing double stranded cDNA from mRNA for use in a sequencing reaction comprising: a) reverse transcribing an isolated mRNA using a first primer hybridized thereto, the first primer comprising a random sequence or poly(dT) at a 3′ end thereof and a first sequence required for a selected sequencing reaction at a 5′ end thereof, thereby producing a first cDNA comprising the first sequence required for the selected sequencing reaction at a 5′ end thereof; b) adding a first 3′ homopolymeric tail to the first cDNA; c) hybridizing a single-stranded oligonucleotide to the 3′ homopolymeric tail of the first strand cDNA, the single-stranded oligonucleotide comprising a 5′ RNA polymerase promoter sequence and a second 3′ homopolymeric tail which is complementary to the first 3′ homopolymeric tail, thereby producing a partially double-stranded DNA with a 5′ single-stranded overhang comprising the RNA polymerase promoter sequence; d) extending the 3′ end of the first cDNA, thereby producing a double stranded RNA polymerase promoter e) transcribing sRNA from the double-stranded RNA polymerase promoter, the sRNA comprising a complement of the first sequence required for the selected sequencing reaction at a 3′ end thereof; f) reverse transcribing the sRNA using a second primer comprising the first sequence required for the selected sequencing reaction at a 3′ end thereof, thereby producing a second cDNA comprising the first sequence required for the selected sequencing reaction at a 5′ end thereof; and g) hybridizing to the second cDNA a third primer comprising a second sequence required for the selected sequencing reaction at a 3′ end thereof and extending the third primer, thereby producing the double-stranded cDNA for use in the sequencing reaction; wherein the single-stranded oligonucleotide comprises SEQ ID NO:3 and the first primer comprises SEQ ID NO:1 or SEQ ID NO:2; or