DNA Amplification, PCR & qPCR

In order to study or detect individual genes or specific DNA regions or mutations of interest, it is often necessary to obtain a large quantity of nucleic acid for study. Rather than isolate a single copy of the target DNA from a large number of cells, it is often more useful to generate multiple copies of a target from a single molecule of DNA or mRNA, via an in vitro amplification method.

As the Polymerase Chain Reaction (PCR) is the most common DNA amplification method in molecular biology, NEB’s product portfolio features a large selection of polymerases geared towards this powerful method. As the first company to sell Taq DNA Polymerase to the research market, the first to discover a PCR-stable, high-fidelity DNA polymerase, and the first to provide reagents for PCR performed in space, NEB has a long history of developing reliable and convenient PCR tools. This commitment has continued with the recent development of OneTaq^® DNA Polymerase for robust routine PCR and Q5^® High-Fidelity DNA Polymerase for robust, ultra high-fidelity PCR (>280 X Taq fidelity). Both product lines have been developed to tolerate a variety of complex templates without experiencing a loss of performance on high-AT or high-GC targets. A variety of NEB polymerases, including OneTaq, Taq and Q5, also benefit from novel aptamer-based hot start technology that does not require a separate activation step.

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.

 

TaqMan® is a registered trademark of Roche Molecular Systems, Inc.
SYBR® is a registered trademark of Molecular Probes, Inc.