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Title: Rapid purification of double-stranded DNA by triple-helix-mediated affinity capture

Abstract

A simple and rapid method for the preparation of highly pure plasmid DNA has been developed. The DNA is directly captured from bacterial cell lysates by formation of a triple-helical structure between the plasmid dsDNA and a 20-base biotinylated oligonucleotide attached to streptavidin-coated magnetic beads and then eluted from the beads in pH 9 buffer solution. No phenol extraction, ethanol precipitation, RNase digestion, or CsCl gradient centrifugation is required. A general purpose cloning vector, pHJ19, was constructed for this application from pUC19 DNA by insertion of a 40-base sequence suitable for triple-helix formation. The approach was also found suitable for the purification of [lambda] bacteriophage DNA. 32 refs., 6 figs., 1 tab.

Authors:
;  [1]
  1. (Univ. of Wisconsin, Madison (United States))
Publication Date:
OSTI Identifier:
6012465
DOE Contract Number:  
FG02-90ER61026
Resource Type:
Journal Article
Resource Relation:
Journal Name: Analytical Chemistry (Washington); (United States); Journal Volume: 65:10
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; DNA; PURIFICATION; AFFINITY; BACTERIA; BACTERIOPHAGES; CHROMOSOMES; DNA-CLONING; MOLECULAR STRUCTURE; CLONING; DNA HYBRIDIZATION; HYBRIDIZATION; MICROORGANISMS; NUCLEIC ACIDS; ORGANIC COMPOUNDS; PARASITES; VIRUSES; 550200* - Biochemistry; 400100 - Analytical & Separations Chemistry

Citation Formats

Ji, H., and Smith, L.M.. Rapid purification of double-stranded DNA by triple-helix-mediated affinity capture. United States: N. p., 1993. Web. doi:10.1021/ac00058a005.
Ji, H., & Smith, L.M.. Rapid purification of double-stranded DNA by triple-helix-mediated affinity capture. United States. doi:10.1021/ac00058a005.
Ji, H., and Smith, L.M.. Sat . "Rapid purification of double-stranded DNA by triple-helix-mediated affinity capture". United States. doi:10.1021/ac00058a005.
@article{osti_6012465,
title = {Rapid purification of double-stranded DNA by triple-helix-mediated affinity capture},
author = {Ji, H. and Smith, L.M.},
abstractNote = {A simple and rapid method for the preparation of highly pure plasmid DNA has been developed. The DNA is directly captured from bacterial cell lysates by formation of a triple-helical structure between the plasmid dsDNA and a 20-base biotinylated oligonucleotide attached to streptavidin-coated magnetic beads and then eluted from the beads in pH 9 buffer solution. No phenol extraction, ethanol precipitation, RNase digestion, or CsCl gradient centrifugation is required. A general purpose cloning vector, pHJ19, was constructed for this application from pUC19 DNA by insertion of a 40-base sequence suitable for triple-helix formation. The approach was also found suitable for the purification of [lambda] bacteriophage DNA. 32 refs., 6 figs., 1 tab.},
doi = {10.1021/ac00058a005},
journal = {Analytical Chemistry (Washington); (United States)},
number = ,
volume = 65:10,
place = {United States},
year = {Sat May 15 00:00:00 EDT 1993},
month = {Sat May 15 00:00:00 EDT 1993}
}