In vitro double transposition for DNA identification.
We present a double transposition technique that inserts two different transposons into target DNA to act as priming sites for amplifying the region between the two transposons for sequencing applications. Unlike some current sequencing approaches, the genome of the unknown target remains intact in this method. The transposition reaction, DNA repair, and subsequent sequencing were performed entirely in vitro, without the need for transformation into bacteria, and resulted in sequence homology with the plasmid DNA target. This approach can reduce the time required for the assay by more than a day compared with standard techniques and reduces the number of required enzymatic steps. In addition, the in vitro method enables transposition to be carried out in automated microfluidic platforms without the need for significant sample manipulation. As a demonstration of incorporating transposition techniques into high-throughput technologies, single transposition reactions were carried out in picoliter-sized droplets generated on a microfluidic platform.
Heredia
Nicholas J
NJ
Lawrence Livermore National Laboratory, Livermore, CA 94550, USA.
Beer
N Reginald
NR
Hara
Christine A
CA
Hiddessen
Amy L
AL
Bailey
Christopher G
CG
D004247:DNA / Q000032:analysis / Q000737:chemistry; D004260:DNA Repair; D004251:DNA Transposable Elements / Q000235:genetics*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D004251:DNA Transposable Elements; D004247:DNA