Thermo Sequenase DNA polymerase and T. acidophilum pyrophosphatase: new thermostable enzymes for DNA sequencing.
A combination of thermostable enzymes has been developed that produces higher quality cycle sequences. Thermo Sequenase DNA polymerase is a thermostable enzyme engineered to catalyze the incorporation of ddNTPs with an efficiency several thousandfold better than other thermostable DNA polymerases. Since the enzyme also catalyzes pyrophosphorolysis at dideoxy termini, a thermostable inorganic pyrophosphatase is needed to remove the pyrophosphate produced during sequencing reactions. Thermoplasma acidophilum inorganic pyrophosphatase (TAP) is thermostable and effective for converting pyrophosphate to orthophosphate. The use of the combination of Thermo Sequenase polymerase and TAP for cycle sequencing yields sequence data with uniform band intensities, allowing the determination of longer, more accurate sequence reads. Uniform band intensities also facilitate interpretation of sequence anomalies and the presence of mixed templates. Sequencing PCR products of DNA amplified from heterozygous diploid individuals results in signals of equal intensity from each allele.
Vander Horn
P B
PB
Amersham Life Science, Cleveland, OH, USA.
Davis
M C
MC
Cunniff
J J
JJ
Ruan
C
C
McArdle
B F
BF
Samols
S B
SB
Szasz
J
J
Hu
G
G
Hujer
K M
KM
Domke
S T
ST
Brummet
S R
SR
Moffett
R B
RB
Fuller
C W
CW
D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015224:Dideoxynucleosides / Q000378:metabolism; D011756:Diphosphates / Q000378:metabolism; D006579:Heterozygote; D006801:Humans; D043564:Inorganic Pyrophosphatase; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011755:Pyrophosphatases / Q000378:metabolism*; D017422:Sequence Analysis, DNA / Q000379:methods*; D019914:Taq Polymerase; D013822:Thermoplasma / Q000201:enzymology*
D015224:Dideoxynucleosides; D011756:Diphosphates; D004247:DNA; D019914:Taq Polymerase; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase