What are the common primer and probe chemistries utilized for qPCR assays?
The types of probes utilized for qPCR fall into 2 classes, those that are not sequence-specific, and those that are sequence-specific. The most popular non-sequence-specific probe chemistry is SYBR Green, which binds to the minor groove of double-stranded DNA and fluoresces 1000-fold more efficiently when bound, than when free in solution. It is the most cost-effective and convenient chemistry for qPCR.
Historically, researchers have worried that SYBR Green chemistry would show inferior specificity when compared to sequence-specific probe chemistries. QIAGEN has eliminated that concern, through the development of complementary SYBR Green-based RT² SYBR Green Mastermixes and RT² qPCR Primer Assays, which are available for any gene in the human, mouse, or rat genome.
Sequence-specific qPCR probe chemistries fall into 2 classes. Bimolecular probes consist of a dual-labeled probe used in combination with flanking forward and reverse primers. TaqMan® and Molecular Beacon probes are popular examples of these chemistries. Unimolecular, sequence-specific probes consist of a dual-labeled hairpin probe which is covalently linked to a forward primer, and is used in combination with an unlabeled reverse primer. Amplifluor and Scorpion probes are popular examples of this class.