Effects of different DNA polymerases in ligation-mediated PCR: enhanced genomic sequencing and in vivo footprinting.
We have developed a simplified procedure for the ligation-mediated polymerase chain reaction (LMPCR) using Thermococcus litoralis DNA polymerase (Vent DNA polymerase). We show that Vent DNA polymerase produces correct, blunt-ended primer extension products with substantially higher efficiency than Thermus aquaticus (Taq) DNA polymerase or modified T7 DNA polymerase (Sequenase). This difference leads to significantly improved genomic sequencing, methylation analysis, and in vivo footprinting with LMPCR. These improvements include representation of all bands with more uniform intensity, clear visualization of previously difficult regions of sequence, and reduction in the occurrence of spurious bands. It also simplifies the use of DNase I cut DNA for LMPCR footprinting.
Garrity
P A
PA
Division of Biology, California Institute of Technology, Pasadena 91125.
Wold
B J
BJ
D000818:Animals; D001419:Bacteria / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000648:ultrastructure; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003850:Deoxyribonuclease I; D007739:L Cells; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D013463:Sulfuric Acid Esters / Q000737:chemistry; D019914:Taq Polymerase; D013698:Templates, Genetic
D004268:DNA-Binding Proteins; D013463:Sulfuric Acid Esters; D004247:DNA; D019914:Taq Polymerase; C034970:bacteriophage T7 induced DNA polymerase; D004253:DNA Nucleotidylexotransferase; D004259:DNA-Directed DNA Polymerase; D003850:Deoxyribonuclease I; C007482:dimethyl sulfate