Patent ID: 8617817

Claim:
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 3â€² end thereof and a first sequence required for a selected sequencing reaction at a 5â€² end thereof, thereby producing a first cDNA comprising the first sequence required for the selected sequencing reaction at a 5â€² end thereof; b) adding a first 3â€² homopolymeric tail to the first cDNA; c) hybridizing a single-stranded oligonucleotide to the 3â€² homopolymeric tail of the first strand cDNA, the single-stranded oligonucleotide comprising a 5â€² RNA polymerase promoter sequence and a second 3â€² homopolymeric tail which is complementary to the first 3â€² homopolymeric tail, thereby producing a partially double-stranded DNA with a 5â€² single-stranded overhang comprising the RNA polymerase promoter sequence; d) extending the 3â€² 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 3â€² end thereof; f) reverse transcribing the sRNA using a second primer comprising the first sequence required for the selected sequencing reaction at a 3â€² end thereof, thereby producing a second cDNA comprising the first sequence required for the selected sequencing reaction at a 5â€² end thereof; and g) hybridizing to the second cDNA a third primer comprising a second sequence required for the selected sequencing reaction at a 3â€² 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 the single-stranded oligonucleotide comprises SEQ ID NO:6, the first primer comprises SEQ ID NO:1 or SEQ ID NO:2, the second primer comprises SEQ ID NO:4 and the third primer comprises SEQ ID NO:7.