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Tech Note: Highly Efficient and Specific Multiplex PCR using TaKaRa Taq DNA Polymerase Hot Start Version

TECH NOTE

Highly Efficient and Specific Multiplex PCR using TaKaRa Taq DNA Polymerase Hot Start Version

The hot start (HS) version of Takara's Taq DNA Polymerase provides higher efficiency and specificity than the standard TaKaRa Taq DNA Polymerase when used in multiplex PCR, with an efficiency comparable to individual amplification reactions.

Overview

Multiplex PCR is a variant of traditional PCR which allows simultaneous amplification of numerous targets using multiple primer pairs in a single amplification reaction. This approach is used in many areas of scientific research, including genotyping applications that require analysis of multiple markers. It is also used in pathogen detection and deletion, mutation, and polymorphism investigation. The technique is demanding, and successful multiplex amplifications require careful primer design, reaction optimization, and a specific, sensitive, and reliable DNA polymerase.

TaKaRa Taq DNA Polymerase Hot Start Version contains a mixture of high-purity TaKaRa Taq DNA Polymerase and a monoclonal antibody to Taq DNA Polymerase, which binds to the polymerase until the temperature is elevated. The binding of this antibody prevents nonspecific amplification due to mispriming and/or formation of primer dimers during reaction assembly. The antibody is then denatured in the initial PCR DNA-denaturation step, releasing the polymerase and allowing DNA synthesis to proceed. Takara HS Taq provides sensitive, reliable amplification, along with reduced background and increased reaction specificity and efficiency.

Comparison with a Standard Taq DNA Polymerase

The performance of Takara HS Taq in a multiplex reaction was compared to its performance in single target reactions, and to that of the standard Takara Taq DNA Polymerase in a multiplex reaction (Figure 1). Takara Taq and Takara HS Taq were each used to amplify a human genomic DNA template with eight different primer pairs, each specific for a target ranging from 84 to 432 bp in size. In addition, Takara HS Taq was used to perform an individual amplification reaction with each of the eight primer pairs.

The sample amplified using Takara HS Taq (Lane 10) shows more efficient multiplex amplification of all the bands individually amplified in Lanes 1–8 than the sample amplified using Takara Taq (Lane 9), and it lacks the low molecular weight non-specific amplification band seen with Takara Taq (Lane 9). The intensities of the bands in Lane 10 are comparable to the intensities of the corresponding individual bands in Lanes 1–8, indicating that Takara HS Taq provides multiplex efficiencies comparable to those observed in individual amplification reactions.

Comparison of TaKaRa Taq Hot Start to standard TaKaRa Taq

Figure 1. Comparing the ability of TaKaRa Taq DNA Polymerase Hot Start Version to amplify human genomic DNA fragments to that of the standard TaKaRa Taq DNA Polymerase. PCR reactions were performed using human genomic DNA as a template and eight different primers for each single fragment. Lanes 1–8 contain individual reactions for each primer pair amplified using Takara HS Taq. Lanes 9 and 10 contain multiplex PCR reactions performed with all eight primer pairs in a single tube, amplified with either the standard Taq DNA Polymerase (Takara Taq) (Lane 9) or Takara HS Taq (Lane 10).

Summary

This experiment demonstrates that multiplex PCR using Takara HS Taq results in target amplification efficiencies equivalent to those of separate (single target) amplification reactions. In addition, Takara HS Taq demonstrates superior efficiency and specificity over standard Taq Polymerase (Takara Taq) in this multiplex PCR application.

Methods

PCR cycling conditions for individual reactions: 1 cycle at 94°C for 30 sec, followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 60 sec.

PCR cycling conditions for multiplex reactions: 1 cycle at 94°C for 30 sec, followed by 30 cycles of 94°C for 30 sec, 57°C for 30 sec, and 72°C for 60 sec, followed by a final step at 72°C for 90 sec.

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