Maximising throughput

By Kamni Vijay, Bio-Rad Laboratories

The polymerase chain reaction (PCR) has traditionally been optimised for specificity and, to a lesser extent, product yield. The speed with which the reaction is completed has been of secondary importance. The availability of software for primer and PCR product design and the use of reagents that can tolerate a range of reaction conditions have allowed researchers to focus on maximising throughput by minimising PCR cycling times. Here we discuss the time savings in fast PCR and fast quantitative PCR (qPCR) that can be made by modifying thermal cycling conditions and choosing appropriate enzymes tailored for fast cycling.

Saving time during PCR and qPCR runs
Standard protocols for amplifying targets of less than 1,000 bp include several steps, each of which can be modified to shorten overall run times from about 90 minutes to less than 30 minutes.

Initial Denaturation – When using an antibody-modified hot-start polymerase, both polymerase activation and initial denaturation can be accomplished in 15-30 seconds at 98°C. 

Denaturation While Cycling – A one second denaturation at 92°C is sufficient for various PCR products. This is consistent with the observation that temperatures above 92°C are unnecessary for denaturing PCR products shorter than 500 bp (Yap and McGee 1991).

Annealing and Extension – Because most polymerases are highly active in the temperature range typical for primer annealing (55-70°C), the annealing and extension steps of a PCR or qPCR protocol can often be consolidated into a single shortened step. A 15 second combined incubation can be sufficient for PCR products up to 500 bp. Furthermore, a combined annealing and extension step at 60°C is typical for qPCR assays using dye-based fluorescence or dual-labeled probes.

Final Extension – A post-PCR final incubation step of five to 10 minutes at 72°C is often recommended to promote complete synthesis of all PCR products, for visualisation on gels or for cloning. We have found that this step can be shortened to 30–60 sec for products up to 1 kb.

Number of Cycles – Target DNA concentration is often unknown in PCR, and may be only a few hundred copies per reaction. For this reason, researchers usually prefer to run 30-45 cycles of PCR despite the potential time savings of running fewer cycles.

Novel enzyme revolutionises thermal cycling
Historically, PCR polymerases provided either high fidelity or high processivity, but not both. Now, using patented Sso7d fusion protein technology, Bio-Rad has incorporated both these parameters into a single enzyme: iProof* high-fidelity DNA polymerase. This novel polymerase accurately amplifies a wide range of DNA templates for use in various applications. Sso7d gives polymerases a sliding grip on the minor groove of the replicated DNA, dramatically increasing processivity without compromising catalytic activity or enzyme stability. This technology improves speed, robustness, capacity of synthesising longer products, and tolerance of PCR inhibitors.

In general, longer targets (above 1 kb) need longer extension times, resulting in runs that can last several hours. The extremely high processivity of iProof polymerase enables extension to be completed more quickly with significantly less enzyme than is required for other polymerases. Time savings of up to three or four fold and increased reaction success have been obtained with iProof DNA polymerase.

Sso7d fusion technology has also been incorporated into a supermix for use in fast real-time qPCR – SsoFast EvaGreen supermix. The unique combination of a fusion DNA polymerase with EvaGreen dye and an optimised buffer system delivers unrivaled speed and performance for various qPCR applications, including high resolution melt analysis and direct PCR. This mix is uniquely tolerant to PCR inhibitors, allowing qPCR results to be obtained in less than 30 minutes.

We have described several ways to maximise the time savings in PCR and qPCR. Portions of this article have been excerpted from Fast PCR: Minimizing Run Times, Maximizing Throughput, appearing in Volume 118 of Bio-Rad’s periodical, BioRadiations. Please visit www.bio-rad.com to download your copy of the full-length article.

[CREDIT TEXT]
* U.S. patent 6,627,424.

Yap EP and McGee JO (1991). Slide PCR: DNA amplification from cell samples on microscopic glass slides. Nucleic Acids Res 19, 4924.

Kamni Vijay, Ph.D., is a Marketing Manager for Genomics at Bio-Rad Laboratories. She directs product development in the areas of PCR, qPCR and emerging technologies. She began her career in the Life Science industry at MJ Research after completing her Ph.D. from University of California, Davis in 2001.