Open AccessCharacterization and modification of phage T7 DNA polymerase for use in DNA sequencing. Final report, June 1, 1988--January 31, 1996
Author(s) -
Charles C. Richardson
Publication year - 1996
Language(s) - English
Resource type - Reports
DOI - 10.2172/491398
Subject(s) - klenow fragment , dna polymerase ii , dna polymerase i , dna polymerase , dna clamp , polymerase , processivity , biology , microbiology and biotechnology , primer (cosmetics) , sequencing by ligation , multiple displacement amplification , taq polymerase , biochemistry , chemistry , exonuclease , dna , genomic library , gene , thermus aquaticus , polymerase chain reaction , reverse transcriptase , dna extraction , organic chemistry , base sequence
This project has focused on the DNA polymerase of phage T7 for use in DNA sequencing. A complex of T7 DNA polymerase and E. coli thioredoxin form a highly processive DNA polymerase. The exonuclease activity of the enzyme can be reduced by chemical or genetic modifications resulting in an enzyme that has several properties useful in sequencing including high processivity and lack of discrimination against dideoxynucleotides. Manganese ion eliminates all discrimination against ddNTPs allowing sequence determination based on band intensity. A single tyrosine residue in the active site of T7 DNA polymerase is responsible for the efficient incorporation of ddNMPs. Replacement of the phenylalanine at this position in Klenow or Taq DNA polymerase with tyrosine eliminates discrimination against ddNTPs, a property that has advantages for cycle sequencing. Pyrophosphorolysis catalyzed by a polymerase results in the hydrolysis of specific fragments in DNA sequencing reactions, a problem that is eliminated by the addition of pyrophosphatase. The thioredoxin domain of gene 5 protein has been identified and transferred to Klenow DNA polymerase to make it processive. We have crystallized a complex of T7 DNA polymerase/thioredoxin bound to a primer-template in the presence of a dNTP