DNA Amplification, PCR & qPCR

For experiments where detection and quantification is required instead of isolation, quantitative PCR (qPCR) uses real-time fluorescence to meaure the amount of a DNA target present at each cycle during a PCR. The most common methods of generating a fluorescent signal are by use of hydrolysis probes (e.g., TaqMan^®), or a double-stranded DNA binding dye, (e.g., SYBR^® Green). Ideally, qPCR master mixes should be evaluated for high reaction efficiency over a wide linear dynamic range, and low variation between repeated reactions across a broad variety of sample types. NEB’s Luna^® product line has been developed to simplify your qPCR reagent selection while accomplishing best-in-class performance.

Despite the ubiquitous nature of PCR and qPCR, it may not be the best option for all amplification needs. For point of care and other diagnostic applications, sequence-specific isothermal amplification methods, that eliminate the need for thermocycling, have been particularly useful. Instead of heat, these methods typically employ a strand-displacing DNA polymerase, like Bst DNA Polymerase, Large Fragment, to separate duplex DNA. To address some of the limitations of current isothermal amplification techniques, NEB has developed the next generation Bst, Bst 2.0 and a WarmStart^® version of this enhanced polymerase, which enables room temperature reaction setup, yet is fully active at temperatures greater than 50°C.

RNA molecules can also be detected and manipulated through amplification via the use of reverse transcriptases (RT), which are RNA-dependent DNA Polymerases. RTs polymerize a strand of DNA that is complimentary to the original RNA template and is referred to as cDNA. This cDNA can then be further amplified through PCR, qPCR or isothermal methods as outlined above or detected in a single reaction using one-step RT-qPCR or RT-LAMP.

Nucleic acid amplification is a foundational process in molecular biology and, as a testament to its utility, new protocols and modifications are being developed constantly. At NEB, our goal is to use our understanding of enzymology, and dedication to providing high-quality products, to offer reagents for all of your applications. Please visit the application pages below to learn more.