High-efficiency DNA ligation for clamp attachment without polymerase chain reaction.
We coupled ligation with mass action to achieve high-efficiency clamp attachment without polymerase chain reaction (PCR). Using a 10-fold molar excess of a GC-rich clamp of synthesized and hybridized oligonucleotides, we achieved the maximum clamp-ligation efficiency in which the clamp was ligated to >95% of 10(10)-10(12) restriction ends of a PCR-amplified fragment. The maximum efficiency was confirmed by ligating the clamp to 10(11)-10(12) restriction ends of human genomic DNA. Our approach can be added to a constant denaturant capillary electrophoresis (CDCE)-based method of analyzing rare point mutants at fractions as low as 10(-6); such mutants appear as small copy numbers in the initial samples. This CDCE-based method alone is applicable to only those DNA sequences juxtaposed with an internally occurring clamp of a higher melting temperature in genomic DNA. Since such sequences represent 9% of the human genome, the addition of clamp ligation significantly increases the scanning range for the human genome without reducing the initial mutant copy numbers. Furthermore, clamp ligation/attachment without PCR prevents PCR-created mutants from interfering with rare mutational analysis. In addition to those applications seeking high-efficiency DNA ligation, our approach can be generally applied to ligation of restriction ends.
Kim
Andrea S
AS
Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. akim@fhcrc.org
Holmquist
Gerald P
GP
Thilly
William G
WG
D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D011088:DNA Ligases / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D016208:Databases, Factual; D019075:Electrophoresis, Capillary / Q000379:methods; D005784:Gene Amplification; D015894:Genome, Human; D006358:Hot Temperature; D006801:Humans; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000737:chemistry; D009994:Osmolar Concentration; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004273:DNA, Neoplasm; D009841:Oligonucleotides; D004247:DNA; D011088:DNA Ligases