Alexandra Isabel Over1, Lawrence Wangh2
1Consultant for Biosearch Technologies, Inc., Petaluma, CA, USA,
2Brandeis University, Department of Biology, Waltham, MA, USA
This presentation introduces a novel ThermaStop™ hot-start reagent, developed in collaboration between Biosearch Technologies, Inc. and Professor Lawrence Wangh’s laboratory at Brandeis University. The ThermaStop hot-start reagent is an oligonucleotide that can adopt a hairpin conformation. This reagent interacts with DNA polymerase in a temperature-dependent manner, rendering the enzyme more selective against 3’ mismatches. As a result, the ThermaStop reagent suppresses the formation of competing, non-specific products, thereby reducing scatter among replicate reactions, and increasing the yield of intended amplicons. ThermaStop hot-start activity rivals or exceeds that of hot-start antibodies. Unlike hot-start antibodies, however, ThermaStop denaturation at high temperatures is reversible. Whenever the reaction temperature is lowered below the primer annealing temperature, the ThermaStop oligo refolds into its active conformation, and thereby provides “cold-stop” protection to amplification products. Thus, the ThermaStop reagent enhances the use of low temperature probes in real-time and end-point asymmetric PCR methods such as LATE-PCR, and also increases the accuracy of downstream applications such as, re-amplification and sequencing. The ThermaStop hot-start reagent is compatible with all PCR strategies because its activity depends only on the functional ratio of reagent to DNA polymerase. The ThermaStop reagent also displays a unique signature in SYBR Green melting curves that can be used to determine the lowest effective concentration of reagent necessary for a particular reaction. In summary, the ThermaStop is a multifaceted, reversible, hot-start reagent that interacts with DNA polymerase to improve the precision and yield of all primer dependent amplification reactions. The ThermaStop reagent is the first of a family of reagents to address all forms of mis-priming that will be commercially available in the near future.
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