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.

What is claimed is: 
     
         1 . An in vitro method for fragmenting DNA, comprising:
 a) forming a plurality of transposon complexes by contacting
 (i) a plurality of modified transposon end sequences, with 
 (ii) a plurality of MuA transposase enzymes, 
 wherein the plurality of modified transposon end sequences are double-stranded nucleic acids having a double-stranded region and a 5′ overhang region, and comprise top and bottom nucleic acid strands, and have MuA R1 and MuA R2 sequences that bind a MuA transposase enzyme, wherein the top and bottom nucleic acid strands contain at least one uracil at different locations within the double-stranded region; 
   b) contacting the plurality of transposon complexes with a plurality of target DNA molecules; and   c) incubating the plurality of transposon complexes and the plurality of target DNA molecules under conditions suitable for transposition of the transposon complexes into the target DNA molecules and for fragmenting the target DNA to produce a plurality of fragmented DNA molecules having both ends joined to the modified transposon end sequence, wherein the modified transposon end sequences that are joined to the fragmented DNA molecules include the at least one uracil.   
     
     
         2 . The method of  claim 1 , further comprising: contacting the plurality of fragmented DNA molecules with uracil DNA glycosylase (UDG) thereby generating a plurality of cleaved fragmented DNA molecules having abasic sites. 
     
     
         3 . The method of  claim 2 , further comprising: contacting the abasic sites with an apurinic/apyrimidinic (AP) endonuclease. 
     
     
         4 . The method of  claim 3 , wherein the apurinic/apyrimidinic (AP) endonuclease comprises an endonuclease IV. 
     
     
         5 . The method of  claim 1 , wherein the plurality of fragmented DNA molecules having both ends joined to the modified transposon end sequence contains a gap on both strands. 
     
     
         6 . The method of  claim 5 , further comprising: filling in the gap on both strands with a DNA polymerase having 5′ to 3′ exonuclease activity of having strand displacement activity. 
     
     
         7 . The method of  claim 2  further comprising: amplifying the cleaved fragmented DNA molecules in an amplification reaction using a plurality of first and second oligonucletide primers having a 3′ portion that is complementary to the transposon end sequence that is retained in the cleaved fragmented DNA molecules, wherein the plurality of first and second primers optionally comprise 5′ adaptor tails that do not hybridize with the cleaved fragmented DNA molecules or with the transposon end sequence. 
     
     
         8 . The method of  claim 7 , wherein the 5′ adaptor tails of the first oligonucleotide primers comprise a tag selected from the group consisting of an amplification tag, a sequencing tag, and a detection tag, and wherein the 5′ adaptor tails of the second oligonucleotide primers comprise a tag selected from the group consisting of an amplification tag, a sequencing tag, and a detection tag. 
     
     
         9 . The method of  claim 8 , wherein the 5′ adaptor tail of the first oligonucleotide primers include a sequence that differs from the 5′ adaptor tail of the second oligonucleotide primers. 
     
     
         10 . The method of  claim 1 , further comprising denaturing the plurality of fragmented DNA molecules to produce a plurality of single-stranded fragmented DNA. 
     
     
         11 . The method of  claim 10 , further comprising immobilizing the plurality of single-stranded fragmented DNA to a support. 
     
     
         12 . The method of  claim 11 , further comprising sequencing the plurality of single-stranded fragmented DNA. 
     
     
         13 . The method of  claim 12 , wherein the sequencing comprises massively parallel sequencing. 
     
     
         14 . The method of  claim 1 , further comprising sequencing the plurality of fragmented DNA molecules. 
     
     
         15 . The method of  claim 15 , wherein the sequencing comprises massively parallel sequencing. 
     
     
         16 . The method of  claim 1 , wherein the top nucleic acid strands comprise the nucleotide sequence according to SEQ ID NO:4 and wherein the bottom nucleic acid strands comprise the nucleotide sequence according to SEQ ID NO:5. 
     
     
         17 . The method of  claim 1 , wherein the 5′ end of the top nucleic acid strands are radioactively labeled.