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Platinum GenoType Tsp DNA Polymerase |
The Invitrogen Platinum GenoType Tsp DNA Polymerase is a recombinant DNA polymerase from a thermophilic bacterial species. Tsp DNA polymerase is a genetically engineered thermostable polymerase (Tsp) for genotyping of dinucleotide repeat loci. Platinum Tsp DNA polymerase is precomplexed with antibodies, which inhibits polymerase activity at room temperature. At approximately 72°C, the antibodies are denatured, and the enzyme is activated, allowing automatic hot-start PCR. Note that the Tsp antibodies are different from those used with Platinum Taq DNA Polymerase.
Tsp DNA polymerase is widely used for genotyping loci with dinucleotide repeats. Determining the size of amplified dinucleotide repeat regions can be challenging since Taq DNA polymerase often adds a non-templated nucleotide, such as one or more adenosines, to PCR products. The fraction of PCR amplicons containing an extra nucleotide is heterogeneous and is dependent on the sequence of the reverse primer used.
Platinum GenoType Tsp DNA polymerase displays reduced activity in adding an extra non-templated nucleotide to PCR products (Figure 1). This enzyme property enables researchers to determine the correct size of the alleles when genotyping loci with dinucleotide repeats by PCR.
Figure 1.Comparison of DNA polymerases in genotyping of dinucleotide repeat markers. Amplification of nga106 locus of Arabidopsis thaliana generated with Taq DNA polymerase (66% n + 1) or Platinum GenoType Tsp DNA Polymerase (0% n + 1).
Reverse primers modified with a 5´-GTGTCTT tail (also PIG-tail primers) are often used with Taq DNA polymerase to enhance the addition of extra nucleotides and to facilitate allele calling [1]. With these modified reverse primers, extra-nucleotide addition was increased but allele calling for the loci tested was not affected for Platinum GenoType Tsp DNA Polymerase. Therefore, Platinum GenoType Tsp DNA Polymerase is effective with either standard or modified reverse primers for PCR genotyping of dinucleotide repeat microsatellites (Table 1).
Standard primer sets | Modified primer sets | |||
---|---|---|---|---|
Tsp DNA Polymerase | Taq DNA Polymerase | Tsp DNA Polymerase | Taq DNA Polymerase | |
Primer sets tested | 81 | 62 | 19 | 19 |
Amplicons with n pattern (no extra nucleotide) | 97% | 2% | 58% | 0% |
Amplicons with n and n+1 pattern (mixed) | 3% | 50% | 42% | 16% |
Amplicons with n+1 pattern (extra nucleotide) | 0% | 48% | 0% | 79% |
The Tsp DNA polymerase is not suitable for amplifying DNA fragments larger than 500 bp. The mutations that eliminate the 5′ exonuclease activity and decrease the extendase activity also diminish the enzyme’s ability to amplify long templates. Table 2 compares the properties of Tsp DNA polymerase and Taq DNA polymerase.
Features | Tsp DNA polymerase | Taq DNA polymerase |
---|---|---|
3′ exonuclease | No | No |
5′ endonuclease | No | Yes |
Extendase activity | Reduced | Yes |
Amplification range | 0.5 kb | 5 kb |
Molecular weight | 70 kDa | 94 kDa |
Reversed primers with 5′-GTGTCTT tail for genotyping | Not required | Recommended |
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For Research Use Only. Not for use in diagnostic procedures.