Compositions and methods for directional nucleic acid amplification and sequencing

Abstract

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.

1 .- 63 . (canceled) 
     
     
         64 . A kit comprising:
 a. one or more primers;   b. a reverse transcription enzyme;   c. a glycosylase; and   d. one or more adaptors wherein one of the adaptors comprises at least one modified dNTP in a ligation strand of the adaptor.   
     
     
         65 . The kit of  claim 64 , further comprising written instructions for use of the kit. 
     
     
         66 . The kit of  claim 64 , further comprising at least one modified dNTP. 
     
     
         67 . The kit of  claim 66 , wherein the modified dNTP comprises dUTP. 
     
     
         68 . The kit of  claim 64 , further comprising reagents for amplification. 
     
     
         69 . The kit of  claim 64 , further comprising reagents for sequencing. 
     
     
         70 . The kit of  claim 69 , wherein the sequencing comprises massively parallel sequencing. 
     
     
         71 . The kit of  claim 64 , further comprising a polyamine. 
     
     
         72 . The kit of  claim 71 , wherein the polyamine comprises N, N-dimethylethylenediamine (DMED). 
     
     
         73 . The kit of  claim 64 , wherein the modified dNTP comprises dUTP. 
     
     
         74 . The kit of  claim 64 , wherein the glycosylase comprises uracil DNA glycosylase (UDG). 
     
     
         75 . The kit of  claim 64 , further comprising an endonuclease. 
     
     
         76 . The kit of  claim 75 , wherein the endonuclease comprises an apurinic/apyrimidinic endonuclease (APE). 
     
     
         77 . The kit of  claim 64 , further comprising one or more polymerases. 
     
     
         78 . The kit of  claim 64 , wherein at least one of the one or more primers comprises a primer comprising a random hybridizing sequence. 
     
     
         79 . The kit of  claim 64 , further comprising dATP, dCTP, dGTP, dTTP, or any mixture thereof. 
     
     
         80 . The kit of  claim 64 , further comprising reagents for end repair.