HotStart DNA polymerases for difficult samples
Hot start DNA polymerase is a modified form of DNA polymerase which avoids a non-specific amplification of DNA by inactivation of the taq polymerase at low temperatures. The polymerase chain reaction (PCR) is a technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. In Hot start PCR, specific antibodies are used to block the Taq-polymerase at lower temperature. An initial step at 95℃ is required for denaturing the antibodies linked to the active center of the enzyme. The anti-Taq antibodies reduce the Taq polymerase activity below 72℃, the optimal temperature at which the enzyme extends the primers. When the specific antibodies detach from Taq-polymerase, the amplification proceeds with greater specificity.
When DNA sequences with a high GC content are amplified, the formation of PCR products can often be compromised by inadequate strand separation and by the propensity for the formation of complex secondary structures. The inability of the DNA polymerase to function through regions of strong secondary structure, such as "hairpins", often leads to the formation of truncated PCR products. In addition, poorly defined amplification products are prevalent when the primers are GC-rich. Despite the inherent challenges, the detection of GC-rich sequences is becoming increasingly important for the molecular diagnosis of hereditary diseases.
We propose DNA polymerases with robust performance in a wide range of models rich in GC and AT. These DNA polymerases have improved tolerance to common PCR inhibitors for crude samples and / or DNA extractions.
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