ABSTRACT
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.
Subject(s)
DNA-Directed DNA Polymerase/metabolism , DNA/chemistry , Pyrophosphatases/metabolism , Sequence Analysis, DNA/methods , Thermoplasma/enzymology , Cloning, Molecular , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , Dideoxynucleosides/metabolism , Diphosphates/metabolism , Heterozygote , Humans , Inorganic Pyrophosphatase , Molecular Sequence Data , Mutagenesis , Taq PolymeraseABSTRACT
SENCAR mice are unusually sensitive to induction of papillomas and squamous cell carcinomas by initiation with 7,12-dimethylbenzanthracene (DMBA) and promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Tumors induced by this protocol were tested for the presence of papillomavirus by immunohistochemistry, Southern blot, reverse Southern blot and dot blot hybridization techniques. Papillomavirus antigens were not detected in any of 235 tumors or 142 non-tumor-bearing skin samples analyzed. Southern blots and dot blots, using a mixed probe of cloned rodent papillomavirus DNA from the multimammate rat, Mastomys natalensis, and the European harvest mouse, Micromys minutus, did not reveal the presence of either episomal or integrated papillomavirus genomes in total cellular DNA extracts from the tumors or non-tumor-bearing skin. To circumvent the possibility that insufficient cross-homology existed to detect a papillomavirus genome with the mixed probe used, DNAs extracted from six papillomas were labeled and each used to probe reference blots that contained 25 cloned papillomavirus genomes excised from their vectors. No evidence for the presence of a papillomavirus genome was detected by this method. Therefore, it is unlikely that papillomaviruses play a role in the induction of tumors in SENCAR mice by two-stage carcinogenesis protocols.
