Purification of Single-Stranded M13 DNA by Cooperative Triple-Helix-Mediated Affinity Capture
✍ Scribed by Arthur F. Johnson; Renfeng Wang; Huamin Ji; Danhua Chen; Richard A. Guilfoyle; Lloyd M. Smith
- Publisher
- Elsevier Science
- Year
- 1996
- Tongue
- English
- Weight
- 352 KB
- Volume
- 234
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
problems in this process may still include yield, purity, A solid-phase triple-helix-mediated affinity capture and multisample handling. This report describes an method is described for the purification of singleautomatable solid-phase approach to M13 DNA isolastranded M13 DNA for use as template in fluorestion based on triple-helix-mediated affinity capture cence-based DNA sequencing reactions. In this which shows prospects of overcoming these bottlemethod, a biotinylated polypyrimidine oligonucleonecks.
tide ''loop'' bound to streptavidin-coated magnetic
Single-stranded DNA template purification is curbeads is used to selectively capture single-stranded rently accomplished by any one of several methods. M13 DNA from high-titer phage supernatant through Examples include polyethylene glycol (PEG) 5 precipitathe formation of a cooperative triple helix (CTH) comtion followed by phenol/chloroform organic extraction plex between the oligonucleotide and a polypurine (1), extraction using a solid-phase medium such as site previously cloned into the M13 vector. Capture is glass (2, 3), diatomaceous earth (4), or an anion-exaccomplished at acidic pH to encourage triple-helix change resin (5) to preferentially bind the DNA, and a formation, while elution is performed at alkaline pH double-helix affinity capture (DAC) method that uses with heating to destroy the CTH complex. The beads an oligonucleotide bound to magnetic streptavidincan be reused up to three times without probe replencoated polystyrene beads to serve as an M13 DNA afishment. Yields of M13 ssDNA in excess of 1 mg per finity capture probe (6, 7). milliliter of culture are obtained, sufficient for use as Recent advances in DNA sequencing technology have template in fluorescence-based DNA sequencing reacaltered the focus and scale of sequencing from a specific tions.