Methods and transposon nucleic acids for generating a dna library

Abstract

A method for the generation of DNA fragmentation library based on a transposition reaction in the presence of a transposon end with an engineered cleaveage site providing facilitated downstream handling of the produced DNA fragments, e.g., in the generation of sequencing templates. Transposon nucleic acids comprising a transposon end sequence and an engineered cleaveage site located in the sequence, e.g., in Mu transposon end sequence, are disclosed.

1 . An in vitro method for generating a DNA library, the method comprising the steps of:
 a) incubating a transposon complex comprising a transposon having a transposon end, and a transposase, with a target DNA of interest under conditions for carrying out a transposition reaction, wherein the transposon end comprises a transposon end sequence which is recognizable by the transposase, and wherein the transposon end sequence comprises at least one modified position, wherein the modified position introduce(s) a cleavage site into the transposon end sequence, and wherein the transposition reaction results in fragmentation of the target DNA and incorporation of the transposon end into the 5′ ends of the fragmented target DNA; and   b) incubating the fragmented target DNA with an enzyme cleaving at the cleavage site so that the transposon ends incorporated to the fragmented target DNA are cleaved at the cleavage site.   
     
     
         2 . The method of  claim 1  further comprising c) amplifying the cleaved, fragmented target DNA in an amplification reaction using a first and second oligonucletide primer complementary to the part of the transposon end retained in the 5′ ends of the cleaved, fragmented target DNA, wherein the first and second primer optionally comprise 5′ adaptor tails. 
     
     
         3 . The method of  claim 1  further comprising the step of contacting the fragments of target DNA obtained from step a) or b) comprising the transposon end at the 5′ ends of the fragmented target DNA with DNA polymerase having 5′-3′ exonuclease or strand displacement activity so that fully double-stranded DNA molecules are produced from the fragments of target DNA. 
     
     
         4 . The method of  claim 2  further comprising the step of denaturating the fully double-stranded DNA molecules to produce single stranded DNA for use in the amplification reaction of step c). 
     
     
         5 . The method of  claim 1 , wherein the transposon end is a Mu transposon end. 
     
     
         6 . The method of  claim 1 , wherein the transposase is MuA transposase. 
     
     
         7 . The method of  claim 1 , wherein the enzyme is an N-glycosylase, an endonuclease, or a restriction enzyme. 
     
     
         8 . The method of  claim 7 , wherein the N-glycosylase is uracil-N-glycosylase. 
     
     
         9 . The method of  claim 7 , wherein the restriction enzyme is a methylation specific restriction enzyme. 
     
     
         10 . The method of  claim 7 , wherein the endonuclease is RNase H. 
     
     
         11 . The method of  claim 1 , wherein the 5′ adaptor tail of the first and/or the second oligonucleotide primers comprise a tag selected from the group consisting of an amplification tag, a sequencing tag, a detection tag, and combinations thereof. 
     
     
         12 . The method of  claim 11 , wherein the adaptor tail of the first and the second oligonucleotide primers comprise a sequencing tag, and the method further comprises denaturating the amplification products obtained from step c) to produce single stranded DNA, annealing the single stranded DNA to a solid support coated with an oligonucleotide complementary to the sequencing tag and performing a DNA sequencing reaction using the single stranded DNA which is immobilized to a solid support as a template. 
     
     
         13 . The method of  claim 1 , wherein the cleavage site in the transposon end sequence is located 15-25 base pairs 5′ direction from the 3′ joining end of the transposon end. 
     
     
         14 . The method of  claim 1 , wherein the cleavage site in the transposon end sequence is located within 25 base pairs 5′ direction from the 3′ joining end. 
     
     
         15 . The method of  claim 1 , wherein the cleavage site in the transposon end sequence is located beyond 25 base pairs 5′ direction from the 3′ joining end. 
     
     
         16 . A modified transposon nucleic acid comprising a transposon end sequence and a joining end, and an engineered cleaveage site located in the transposon end sequence. 
     
     
         17 . The modified transposon nucleic acid of  claim 16  wherein the engineered cleavage site is located 15-25 base pairs 5′ direction from the 3′ joining end of the transposon end. 
     
     
         18 . The modified transposon nucleic acid of  claim 16 , wherein the transposon end sequence is Mu transposon end sequence. 
     
     
         19 . The modified transposon nucleic acid of  claim 16 , wherein the cleavage site is an uracil nucleic acid base, a plurality of ribonucleic acid bases, or a methylated nucleic acid base introduced into the transposon end sequence. 
     
     
         20 . The modified transposon nucleic acid of  claim 16 , wherein the cleavage site is a restriction enzyme site. 
     
     
         21 . The modified transposon nucleic acid of  claim 16 , further comprising a transposase and optionally DNA primers complementary to a region of the modified transposon. 
     
     
         22 . A method comprising use of the transposon nucleic acid according to  claim 16  for generating a DNA library or DNA sequencing templates. 
     
     
         23 . A kit comprising
 a transposase,   a transposon, wherein the transposase binds the transposon, and wherein the transposon is modified to include a cleavage site.   a first and second primer complimentary to a region of the transposon,   at least one additional component selected from the group consisting of a buffer, a polymerization enzyme, an N-glycosylase, an endonuclease, and a restriction enzyme, and instructions for forming a DNA library from a target DNA.