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Figure 1: Amplification of a 131 bp fragment of the human TNF gene using HotMaster and competing Hot Start technologies.
Lanes 1-3: Standard Taq
Lanes 4-6: Eppendorf HotMaster Taq
Lanes 7-9: Chemically blocked Taq
Lanes 10-12: Antibody-blocked Taq
M: 100 bp ladder (NEB)

Processivity:

HotMaster Taq can be used to amplify fragments up to 5.0 kb long. HotMaster easily amplifies a 5.0 kb fragment from Lambda Phage. In contrast, a chemically modified Hot Start enzyme barely produces any amplification of this same fragment (Fig. 2).

Figure 2: Amplification of a 5.0 kb Lambda DNA target using Eppendorf HotMaster Taq and Competitor A Hot Start Taq.
1 l of the PCR reactions was loaded onto a 1% agarose gel. Replicates are shown for each enzyme.

Sensitivity

To test the limits of the amount of template that can be amplified in a highly complex background, a fragment of the male-specific human single-copy gene SRY was amplified using HotMaster Taq. Based on the knowledge that 6 pg of human gDNA equals one copy of the genome, serial dilutions of male DNA were spiked into a constant background of 100 ng female DNA so that the PCR reactions contained 0, <10, 10, 100, 1,000 or 10,000 copies of the target gene.The data show (Fig. 3) that HotMaster Taq DNA Polymerase is able to amplify even extremely low (<10) target DNA molecules in a high background of non-specific DNA.

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