"Hot-Start" PCR products | Lyophilized Mastermixes (rt-PCR, qPCR) | light-Cycler Capillaries |
for q-PCR and "difficult" PCR | for "hot-start" PCR (freeze dried, blue stain | inexpensive alternativ |
for "hot-start" PCR, red stain | for qPCR (freeze dried, no stain) | incl. rotoradapter |
for Real Time and qPCR | ||
for Real Time PCR with carry-over-protection | ||
PCR beginner Kit | ||
Hot start Mastermix with reference dye | ||
for effective one-Tube RT PCR |
What´s "Hot Start" PCR?
<em>Taq</em> polymerase is almost as efficient as Klenow polymerase at 37oC; consequently, if primers miss-anneal at low temperature prior to initial template denaturation, "non-specific" amplification may occur. This may be avoided by only adding enzyme after the initial denaturation, before the reaction cools to the chosen annealing temperature to avoid mixing of primers and target DNA at low temperatures in the presence of Taq polymerase.
The method to add the enzyme to the reaction tube which is standing in the PCR machine is a common procedure, but the disadvantage is the handling procedure with the risk of e.g. cross contaminations. Such competing side reactions as mispriming and primer dimerisation can significantly affect the sensitivity of the PCR reaction (Chou et al. 1992). Hence there has been a clear trend towards the use of thermostable polymerases that require heat activation prior to the PCR reaction (SA Bustin, 2002), a so called "Hot Start".
Hot Start enzymes
These enzymes are supplied in an inactive state, which shows no polymerase activity at ambient temperatures. This prevents: elongation of unspezific annealed primers or formation of primer dimers at low temperatures during PCR setup and the initial PCR cycle.
Hot Start DNA polymerases are activated by incubation at 95°C during the first, initial denaturation step in your existing PCR-cycler program. In principle there are two kinds of hot start enzymes existing:
1. chemically modified polymerases which require a longer first initial denaturation step for activation.
2. polymerase blocked by specific antibodies, which allows normal setup protocol and procedure.
Bioron recommends to use SuperHot Taq Cat # 119010 for Real Time PCR and for all other applications where Hot Start is a crucial point. Current real-time chemistries use separate PCR primers and probes, and the generation of a fluorescent signal depends on intermolecular interactions between template, primers and probes. Hot start enzymes are available from all major suppliers and despite their claims, there is probably little difference in the real-life performance of these enzymes (SA Bustin, 2002).
Bioron´s SuperHot Taq, an antibody inhibited hot start enzyme was successful tested in Taqman RealTime PCR.
Literature
Chou Q, Russell M, Birch DE, Raymond J & Bloch W 1992; Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. <em>Nucleic Acids Research 20</em>, 1717-1723.;
SA Bustin; <em>Journal of Molecular Endocrinology</em> (2002) 29, 23-39)
