Pb. Vanderhorn et al., THERMO SEQUENASE(TM) DNA-POLYMERASE AND T-ACIDOPHILUM PYROPHOSPHATASE- NEW THERMOSTABLE ENZYMES FOR DNA-SEQUENCING, BioTechniques, 22(4), 1997, pp. 758
A combination of thermostable enzymes has been developed that produces
higher qualify Cycle sequences. Thermo Sequenase(TM) DNA polymerase i
s a thermostable enzyme engineered to catalyze the incorporation of dd
NTPs with an efficiency several thousandfold better than other thermos
table DNA polymerases. Since the enzyme also catalyzes pyrophosphoroly
sis at dideoxy termini, a thermostable inorganic pyrophosphatase is ne
eded to remove the pyrophosphate produced during sequencing reactions.
Thermoplasma acidophilum inorganic pyrophosphatase (TAP) is thermosta
ble and effective for converting pyrophosphate to orthophosphate. The
use of the combination of Thermo Sequenase polymerase and TAP for cycl
e sequencing yields sequence data with uniform band intensities, allow
ing the determination of longer; more accurate sequence reads. Uniform
band intensifies also facilitate interpretation of sequence anomalies
and the presence of mixed templates. Sequencing PCR products of DNA a
mplified from heterozygous diploid individuals results in signals of e
qual intensity from each allele.