Open AccessEffects of different DNA polymerases in ligation-mediated PCR: enhanced genomic sequencing and in vivo footprinting.
Author(s) -
Paul Garrity,
B Wold
Publication year - 1992
Publication title -
proceedings of the national academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.89.3.1021
Subject(s) - hot start pcr , taq polymerase , sequencing by ligation , dna polymerase , biology , polymerase , inverse polymerase chain reaction , thermus aquaticus , dna polymerase ii , microbiology and biotechnology , primer (cosmetics) , dna nanoball sequencing , multiple displacement amplification , primer dimer , polymerase chain reaction , genetics , dna , chemistry , nested polymerase chain reaction , genomic library , gene , multiplex polymerase chain reaction , reverse transcriptase , dna extraction , base sequence , organic chemistry
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.
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