Transposon nucleic acids comprising a calibration sequence for dna sequencing

Abstract

Transposon nucleic acids comprising a transposon end sequence and a calibration sequence for DNA sequencing in the transposon end sequence. In one embodiment, the transposon end sequence is a Mu transposon end. A method for the generation of DNA fragmentation library based on a transposition reaction in the presence of a transposon end with the calibration sequence providing facilitated downstream handling of the produced DNA fragments, e.g., in the generation of sequencing templates.

What is claimed: 
     
         1 . A nucleic acid comprising a modified transposon end sequence, wherein
 (a) the modified transposon end sequence comprises (i) a first strand having the nucleotide sequence of SEQ ID NO: 1 that is modified to contain a calibration sequence that is four nucleotides in length and (ii) a second strand that is complementary to the first strand, and   (b) the calibration sequence contains four different nucleotide bases in any order and is located in the 3′ end region of sequence of SEQ ID NO: 1 3′ of position 17 of SEQ ID NO: 1.   
     
     
         2 . The nucleic acid of  claim 1 , wherein the calibration sequence is chosen from among the sequences of 5′-TCAG-3′, 5′-GTCA-3′ and 5′-TGCA-3′. 
     
     
         3 . The nucleic acid of  claim 1 , wherein the modified transposon end sequence comprises the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 6. 
     
     
         4 . The nucleic acid of  claim 1 , wherein the modified transposon end sequence comprises a cleavage site. 
     
     
         5 . An in vitro transpososome assembly reaction mixture comprising a transposase, a nucleic acid comprising a modified transposon end sequence, and reaction mixture components suitable for assembly of transposoomes, wherein
 (a) the modified transposon end sequence comprises (i) a first strand having the nucleotide sequence of SEQ ID NO: 1 that is modified to contain a calibration sequence that is four nucleotides in length and (ii) a second strand that is complementary to the first strand,   (b) the calibration sequence contains four different nucleotide bases in any order and is located in the 3′ end region of sequence of SEQ ID NO: 1 3′ of position 17 of SEQ ID NO: 1 and   (c) the modified transposon end sequence and the transposase are capable of forming a transpososome.   
     
     
         6 . The in vitro transpososome assembly reaction mixture of  claim 5 , wherein the calibration sequence is chosen from among the sequences of 5′-TCAG-3′, 5′-GTCA-3′ and 5′-TGCA-3′. 
     
     
         7 . The in vitro transpososome assembly reaction mixture of  claim 5 , wherein the modified transposon end sequence comprises the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. 
     
     
         8 . The in vitro transpososome assembly reaction mixture of  claim 5 , wherein the modified transposon end sequence comprises the nucleotide sequence of SEQ ID NO: 5. 
     
     
         9 . A kit comprising a nucleic acid comprising a modified transposon end sequence of  claim 1 , a transposase and a buffer for performing a transposition reaction. 
     
     
         10 . The kit of  claim 9 , further comprising a DNA polymerase. 
     
     
         11 . A method for in vitro assembly of one or more transpososome complexes, comprising contacting a transposase and a nucleic acid comprising a modified transposon end sequence under a condition for forming a transpososome complex, thereby forming one or more transpososome complexes, wherein
 (a) the modified transposon end sequence comprises (i) a first strand having the nucleotide sequence of SEQ ID NO: 1 that is modified to contain a calibration sequence that is four nucleotides in length and (ii) a second strand that is complementary to the first strand, and   (b) the calibration sequence contains four different nucleotide bases in any order and is located in the 3′ end region of sequence of SEQ ID NO: 1 3′ of position 17 of SEQ ID NO: 1.   
     
     
         12 . The method of  claim 11 , wherein the calibration sequence is chosen from among the sequences of 5′-TCAG-3′, 5′-GTCA-3′ and 5′-TGCA-3′. 
     
     
         13 . The method of  claim 11 , wherein the modified transposon end sequence comprises the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 6. 
     
     
         14 . The method of  claim 11 , wherein the modified transposon end sequence comprises a cleavage site. 
     
     
         15 . The method of  claim 13 , further comprising contacting the one or more transpososome complexes with a plurality of target nucleic acids. 
     
     
         16 . The method of  claim 15 , further comprising incubating the one or more transpososome complexes and plurality of target nucleic acids under a condition for performing a transposition reaction, wherein the transposition reaction results in fragmentation of the plurality of target nucleic acids and incorporation of a modified transposon end sequence into the ends of the fragmented target nucleic acids, thereby generating a plurality of target nucleic acid fragments attached at both ends to a modified transposon end sequence. 
     
     
         17 . The method of  claim 16 , further comprising contacting a DNA polymerase having 5′-3′ exonuclease or strand displacement activity with the plurality of target nucleic acid fragments to generate fully double-stranded nucleic acid molecules. 
     
     
         18 . The method of  claim 17 , further comprising denaturating the fully double-stranded nucleic acid molecules to produce single-stranded nucleic acids, and amplifying the single-stranded nucleic acids. 
     
     
         19 . The method of  claim 18 , further comprising sequencing the single-stranded nucleic acids.