Platinum Genotype Tsp DNA Polymerase

The Invitrogen Platinum GenoType Tsp DNA Polymerase is a recombinant DNA polymerase from a thermophilic bacterial species. It is a genetically engineered thermostable polymerase (Tsp) for genotyping of dinucleotide repeat loci. The enzyme is complexed with a proprietary antibody to inhibit polymerase activity at room temperature.

Highlights

  • Improved accuracy of allele size determination—Exhibits minimal nontemplated nucleotide addition (also known as extendase activity); lacks both 5′ and 3′ exonuclease activities
  • Convenient workflow—Can substitute for Taq DNA polymerase in PCR and allows room-temperature setup
  • Increased specificity—Precomplexed with anti-Tsp antibodies for use in hot-start PCR

Tsp DNA polymerase for genotyping loci with dinucleotide repeats

Determining the size of amplified dinucleotide repeat regions can be challenging since Taq DNA polymerase often adds a nontemplated nucleotide to PCR products (e.g., one or more adenosines). The fraction of PCR amplicons containing an extra nucleotide is heterogenous and is dependent on sequence of the reverse primer used.

Platinum Genotype Tsp DNA polymerase displays reduced activity in adding an extra nontemplated nucleotide to PCR products. This enzyme property enables researchers to determine the correct size of the alleles when genotyping loci with dinucleotide repeats by PCR.

Comparison of DNA polymerases in genotyping of dinucleotide repeat markers
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 addition of extra nucleotides and to facilitate allele calling [1]. With these modified reverse primers, extranucleotide 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.

  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%

Properties of Tsp DNA polymerase vs. Taq DNA polymerase

The following table compares 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

Amplification range of Tsp DNA polymerase

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.


Tsp antibody properties

Platinum Tsp DNA polymerase is precomplexed with antibodies, which are denatured at about 72°C. Denaturation of the antibodies in the first step of PCR activates the enzyme and warrants automatic hot-start PCR. Note that the Tsp antibodies are different from those used with Platinum Taq DNA Polymerase.


Ordering information


References

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Product manual

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