Compositions and methods for directional nucleic acid amplification and sequencing

摘要

The invention provides methods and compositions, including kits, for directional nucleic acid amplification and sequencing. The invention further provides methods and compositions for the construction of directional cDNA libraries.

主权项

1. A method for construction of a directional cDNA library, the method comprising:
a. reverse transcribing an RNA sample to generate a first strand cDNA; b. generating a second strand cDNA from the first strand cDNA, wherein the generating the second strand cDNA comprises incorporating a modified dNTP during the generating the second strand cDNA, synthesis and incorporated into the second strand, thereby generating a double-stranded cDNA; c. performing end repair on the double-stranded cDNA; d. ligating two adaptors to the double-stranded cDNA, wherein each of the two adaptors comprise a ligation strand and a non-ligation strand, wherein only one of the two adaptors has the modified dNTP incorporated into the ligation strand of the adaptor, wherein the ligation strand of each of the two adaptors is configured to ligate to a 5′ end of the double-stranded cDNA; e. extending each 3′ end of the double-stranded cDNA with a polymerase, wherein the ligation strand of one of the two adaptors is used as a template; and f. selectively cleaving the second strand cDNA and the adaptor that has the modified dNTP using a cleavage agent, thereby generating a directional cDNA library comprising the first strand cDNA.