Modified transposases for improved insertion sequence bias and increased DNA input tolerance

Abstract

Presented herein are transposase enzymes and reaction conditions for improved fragmentation and tagging of nucleic acid samples, in particular altered transposases and reaction conditions which exhibit improved insertion sequence bias, as well as methods and kits using the same.

What is claimed is: 
     
       1. A fusion protein comprising a mutant Tn5 transposase having transposase activity and a polypeptide fusion domain, wherein the fusion protein comprises SEQ ID NO: 25. 
     
     
       2. A transposome complex comprising the fusion protein of  claim 1  and a polynucleotide comprising a transposon end. 
     
     
       3. The transposome complex of  claim 2 , wherein the polynucleotide further comprises a tag. 
     
     
       4. A kit for performing an in vitro transposition reaction comprising the transposome complex of  claim 2 . 
     
     
       5. The transposome complex of  claim 3 , wherein the tag comprises a sequencing tag domain. 
     
     
       6. A method of making the fusion protein of  claim 1  comprising culturing a host cell transformed with the nucleic acid of SEQ ID NO: 24, wherein said host cell expresses said nucleic acid. 
     
     
       7. A method for in vitro transposition comprising contacting the transposome complex of  claim 2  with a target DNA. 
     
     
       8. The method of  claim 7 , wherein the polynucleotide comprising the transposon end further comprises a first tag; wherein said polynucleotide comprises two strands; wherein (i) one of the strands is a transferred strand that comprises the first tag at the 5′ end of the transposon end, and (ii) the other strand is a non-transferred strand which is complementary to the transposon end of the transferred strand. 
     
     
       9. The method of  claim 8 , wherein the contacting comprises fragmenting the target DNA and joining the transferred strand to the 5′ end of the fragments of the target DNA generated by said fragmenting, thereby generating a population of 5′-tagged DNA fragments comprising the first tag on the 5′ end; and wherein the method further comprises joining a second tag to the 3′ end of the 5′-tagged DNA fragments to generate a library of di-tagged DNA fragments. 
     
     
       10. The method of  claim 7 , wherein the polynucleotide comprising the transposon end further comprises a first tag that comprises a sequencing tag domain, wherein the first tag is at the 5′ end of the transposon end; wherein the contacting occurs under conditions whereby the target DNA is fragmented, and the transposon end of the polynucleotide is transferred to the 5′ ends of the DNA fragments, thereby producing double-stranded DNA fragments wherein the 5′ ends of said DNA fragments are tagged with the first tag and the 3′ ends of said DNA fragments have a single-stranded gap. 
     
     
       11. The method of  claim 10 , wherein the method further comprises (i) incubating the double-stranded DNA fragments with a nucleic acid-modifying enzyme under conditions whereby a second tag is attached to the 3′ ends of the double-stranded DNA fragments to form di-tagged DNA fragments, (ii) hybridizing sequencing primers comprising a portion corresponding to the sequencing tag domain; (iii) extending said sequencing primers; and (iv) detecting the identity of nucleotides adjacent to the sequencing tag domain of the di-tagged DNA fragments; wherein said method optionally comprises amplifying the di-tagged DNA fragments prior to step (ii) by providing a polymerase and an amplification primer corresponding to a portion of the polynucleotide comprising the transposon end and the first tag.