In order to study individual genes or specific DNA regions 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 and the first to discover a PCR-stable, high-fidelity DNA polymerase, NEB has a long history of developing reliable and convenient PCR tools. This commitment has continued with the recent development of OneTaq® for robust routine PCR and Q5® High-Fidelity DNA Polymerase for robust, ultra high-fidelity PCR (>100 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.
This overview will walk you through how the Polymerase Chain Reaction (PCR) works.
NEB offers a variety of DNA polymerases for all of your experimental needs. High-fidelity, multiplex, and fast PCR are just a few of the types of amplification reactions you may need to conduct, and Breton explains which polymerases to choose.
Why do primer melting temperatures matter? How is an annealing temperature calculated? Nicole has answers to these questions & more, in this quick video!
Despite the ubiquitous nature of PCR, 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 set up, 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 or isothermal methods as outlined above.
Nucleic acid amplification is a foundational process in molecular biology and, as a testament to it’s 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.
FAQs for DNA Amplification & PCR
Protocols for DNA Amplification & PCR