Method for Differentiation of Polynucleotide Strands

Abstract

Objective of the present invention is to provide a method for keeping of directional information in double-stranded DNA. We suggest to convert polynucleotide into a hybrid double-stranded DNA. One particular strand of this hybrid double-stranded DNA should be synthesised using at least one modified nucleotide. Thus, this particular strand would contain modified nucleotides along the whole length. Density of directional markers would not depend on the length of polynucleotides. Any internal fragments of the hybrid double-stranded DNA would have directional information. When it is necessary the modified strand may be easily degraded or separated from the other strand. It was found that such hybrid double-stranded DNA may be easily generated in a number of molecular biology tasks and may be used for molecular cloning, library preparation and strand separation.

1 - 16 . (canceled) 
     
     
         17 . A kit for preparing a nucleic acid for strand-specific sequencing, comprising:
 (a) an RNA-dependent DNA polymerase for producing first strand cDNA from an RNA sample   (b) a primer for priming the first strand cDNA;   (c) a modified nucleotide;   (d) a non-thermostable DNA-dependent DNA polymerase for copying the first strand cDNA to produce second strand cDNA;   (e) an optional glycosylase; and   (f) a thermostable, non-strand-displacing polymerase for amplifying, by PCR, at least part of the first strand cDNA but not the second strand cDNA,   wherein: if the thermostable, non-strand displacing polymerase of (f) is suppressed by a template comprising the modified nucleotide then the kit does not comprise the glycosylase.   
     
     
         18 . The kit of  claim 17 , wherein the kit further comprises dATP, dTTP, dCTP and dGTP. 
     
     
         19 . The kit of  claim 17 , wherein the modified nucleotide of (c) is dUTP and the glycosylase of (e) is uracil-N-glycosylase that removes uracil from the second strand. 
     
     
         20 . The kit of  claim 17 , wherein the kit contains glycosylase of (e). 
     
     
         21 . The kit of  claim 17 , wherein the thermostable, non-strand displacing polymerase of (f) is suppressed by a template comprising modified nucleotide and the kit does not comprise the glycosylase. 
     
     
         22 . The kit of  claim 21 , wherein the polymerase of (f) is Phusion polymerase or a mutant thereof. 
     
     
         23 . The kit of  claim 17 , wherein the primer of (b) is an oligo-dT primer. 
     
     
         24 . The kit of  claim 17 , wherein the primer of (b) is random primer 
     
     
         25 . The kit of  claim 17 , wherein the RNA-dependent DNA polymerase is HIV-1 reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, or a mutant thereof. 
     
     
         26 . The kit of  claim 17 , wherein the polymerase of (d) is DNA polymerase I or a mutant thereof. 
     
     
         27 . The kit of  claim 17 , wherein the polymerase of (f) is Taq polymerase, Tth polymerase, Pfu polymerase or Phusion, or a mutant thereof. 
     
     
         28 . The kit of  claim 17 , further comprising beads for purifying poly-A +  RNA. 
     
     
         29 . The kit of  claim 17 , further comprising a matrix for removing nucleotides between first strand DNA synthesis and second strand DNA synthesis. 
     
     
         30 . The kit of  claim 17 , further comprising an inhibitor of DNA-dependent, but not RNA-dependent, DNA synthesis. 
     
     
         31 . The kit of  claim 30 , wherein the inhibitor is actinomycin D.