DNA聚合酶

Glycerol is used with most enzymes as a cryoprotectant and stabilizer. The presence of hydroxyl groups disrupts the hydrogen bonding between water molecules and preventing the formation of ice crystals during freezing that can damage enzymes. A composition containing 50% glycerol having a freezing point to as low as -22˚C.

Glycerol tends to persist as a plasticizer or excluded liquid layer during drying – impeding the drying process, concentrations above 0.1% are generally incompatible with lyophilization.

Highly concentrations help 1/ reduce volumes when drying down, making the process faster, 2/ reduces volumes in microfluidic and cartridges, 3/ increases sample volume, 4/ facilitates optimization, 5/ speed up the PCR reaction (as long as there is substrate available to bind to), 6/ increases sensitivity and yield and 7/ increased tolerant to PCR inhibitors. As a result, they can be employed in a wide range of assay formats, allowing more flexibility for development and cost saving during commercialization.

These terms refer to parameters to be considered when performing when testing PCR enzymes. Yield: The amount of DNA produced in a PCR reaction. High yield increases sensitivity. Fidelity: The accuracy of the enzyme at incorporating the correct dNTP to the elongating DNA strand. For standard PCR this is not important, but for sequencing or expression a high-fidelity may be required. Processivity: The length of time a polymerase is associated with the template and therefore the size of fragment which can be amplified (the amplicon). Specificity: A measure of the unwanted by-products generated in a reaction. This is the reason for having hot start PCR, this prevents miss-priming at low temperatures and non-specific amplification.

With an antibody hot start DNA polymerase, the polymerase is bound by antibodies at their active sites to block enzyme activity at room temperature. Advantages 1/ the polymerase features similar as the non–hot start version, since antibodies do not alter the polymerase 2/ Short activation time as the initial denaturation step of PCR activates the polymerase 3/Full enzyme activity restored after activation. Considerations 1/ Higher level of exogenous proteins (i.e., antibodies) present in the reaction 2/ Antibodies may be of animal origin, leading to additional bioburden.

With a chemical hot start DNA polymerase, the polymerase is covalently linked with chemical groups to block enzyme activity at room temperature. Advantages 1/ More stringent than antibody hot-start methods, so there is no activity even for long periods at room temperature, minimizes non-specific amplification 2/ Low bioburden, as it is free of animal and bacterial-origin components Considerations 1/ Longer activation time required for the polymerase to become fully active.

Aptamers are engineered oligonucleotides that bind to the active site of the high-fidelity DNA polymerase but are denatured and released as the temperature is increased during the PCR amplification cycle. This aptamer-mediated inhibition/activation process is however fully reversible, and so at the end of thermal cycling, when the temperature of the reaction is decreased, the aptamer refolds and rebinds to high-fidelity DNA polymerase, inhibiting any further activity in the sample. This has proven to be important in workflows where undesired polymerase activity after reaction completion can disturb baseline readings, such as NGS following amplification of a library.