Abstract
This article describes the sequence-specific isolation and purification of intact double-stranded DNA (dsDNA) by oligonucleotide/PNA-assisted affinity capture (OPAC). The OPAC assay is based on selective tagging of a DNA duplex by biotinylated oligodeoxyribonucleotide (ODN) through formation of a so-called PD-loop. The PD-loop is assembled with the aid of a pair of PNA "openers", which allow sequence-specific targeting with a Watson-Crick complementary ODN probe in the exposed region of the dsDNA. The protocol involves three steps. First, two cationic bis-PNAs locally pry the DNA duplex apart at a predetermined site. Then, the exposed DNA single strand is targeted by a complementary biotinylated ODN to selectively form a stable PD-loop complex. Finally, the capture of dsDNA is performed using streptavidin covered magnetic beads. The OPAC procedure has many advantages in the isolation of highly purified native DNA over other affinity capture and amplification techniques.
Highlights
Methods for purification of specific double-stranded DNA (dsDNA) molecules from heterogeneous DNA mixtures or directly from biological samples have contributed immensely to molecular biology, genomics and the emerging field of DNA diagnostics
Point mutations are often introduced during PCR amplification and information about post-synthetic modifications of DNA nucleotides is lost
If a single round of ODN/PNA-assisted affinity capture (OPAC) is enough for dsDNA selection or when it is necessary to remove bound PNA from DNA for subsequent manipulation with captured materials, high-salt buffer D should be used at the end of the procedure
Summary
Methods for purification of specific dsDNA molecules from heterogeneous DNA mixtures or directly from biological samples have contributed immensely to molecular biology, genomics and the emerging field of DNA diagnostics. When a pair of such PNA “openers” bind to closely located homopurine DNA tracts separated by any short sequence of nucleobases, the two adjacent P-loops merge and an extended open region emerges inside dsDNA. Affinity capture of the targeted dsDNA fragments is possible by employing ODN probes with biotin or other tags that allow convenient capture.
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