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

Many fluorescent qPCR primer- and probe-based chemistries have been devised and are available from different commercial vendors for molecular diagnostic tests, the two most commonly used are:
• Hydrolysis (TaqMan) probes. The main advantages of using hydrolysis probes are high specificity, a high signal-to-noise ratio, and the ability to perform multiplex qPCR reactions. The disadvantages are that the initial cost of the probe.
• Molecular beacons. Molecular beacons are highly specific, can be used for multiplexing, and if the target sequence does not match the beacon sequence exactly, hybridization and fluorescence will not occur. Unlike hydrolysis probes, molecular beacons are displaced but not destroyed during amplification and can be used for melt curve analysis if necessary. The main disadvantage is that they are difficult to design, requiring a stable hairpin stem that is strong enough that the molecule will not spontaneously fold into non-hairpin conformations but not be too strong, or it may not properly hybridize to the target.

The Low DNA qPCR Mix has some inhibitor tolerance, but for amplification from crude lysates or inhibitor-rich samples we have the universal Inhibitor-Tolerant qPCR mix and for even greater sensitivity, we have the sample specific (blood, saliva, urine, stool, and plant) mixes.

Genotyping refers to the process of determining genetic variations among individuals in a population. Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation and are single-base differences at a specific locus. These genotype changes can confer positive or negative phenotypic outcomes, such as more robust stress tolerance in plants or enhance the susceptibility for disease in humans. Hence, SNPs are often useful markers for understanding the biology of organisms. Genotyping is done with two allele-specific probes that have a single base mismatch and a pair of primers at each end of the SNP of interest. Depending on which base is present at the SNP position, the probe will either not hybridize correctly during the qPCR and be left intact or hybridize correctly and be cleaved to give a signal. Since the dual-labeled probe assay is based on PCR, it is relatively simple to implement, and the scale can be drastically increased by performing many simultaneous reactions together.

Genotyping also refers to copy number variation (CNV), which results from having different numbers of copies of a DNA segment in various genomes. Where the copy number variation is for an encoded gene, this can lead to susceptibility or resistance to disease. This uses two sets of primers and probes, one for a calibrator gene that does not change and the other for the gene thought to have the changed copy number. A comparison to the Ct values of each allows you to determine if the copy number has changed and by how much.