Uniform 13C/15N-labeling of DNA by tandem repeat amplification.
An optimized procedure has been described for the large-scale production of stable isotopeenriched duplex oligonucleotides of designed sequence. Large-scale production of labeled nucleotide triphosphates can be produced in this procedure simultaneously with labeled proteins, thereby providing synthetic dNMP precursors at no additional cost. The procedure is robust, with a minimum product:template yield of 800:1 overall, and produces > 99% single-length product. Tandem repeat PCR amplification is a general approach to large scale synthesis of duplex oligonucleotides and may have applications to both NMR and X-ray methods, particularly for product lengths in excess of 25 base pairs where failed sequences from solid-phase synthesis can be difficult to remove chromatographically. A drawback of the present approach is that the product is a duplex of two equal-length strands, making single-stranded products more difficult to prepare. For this reason, it could be preferable to produce single-stranded products by the [figure: see text] method of Zimmer and Crothers. Although a single base type can be selectively enriched in this approach, chemical synthesis will provide greater flexibility for labeled DNAs requiring site-specific labels at only one or a small number of nucleotide positions in the sequence. Therefore, maximum flexibility in labeling patterns can be realized by judicious choice of labeling method appropriate to the type of DNA product and extent of isotopic enrichment desired.
Werner
M H
MH
Laboratory of Molecular Biophysics, Rockefeller University, New York, New York 10021, USA.
Gupta
V
V
Lambert
L J
LJ
Nagata
T
T
D001483:Base Sequence; D002247:Carbon Isotopes; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D003903:Deuterium; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D005784:Gene Amplification; D046911:Macromolecular Substances; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D016133:Polymerase Chain Reaction / Q000379:methods; D020080:Tandem Repeat Sequences
D002247:Carbon Isotopes; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D009587:Nitrogen Isotopes; D003903:Deuterium