Press Releases: A robust method for Direct PCR from whole blood

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Tuesday, 4th December 2007 

Efficient PCR from whole blood using Finnzymes High Performance PCR solution

Finnzymes have developed a range of reagents, consumables and instrumentation to allow fast PCR in inhibiting conditions. Here we describe an application for amplifying exogenous and human genomic DNA from whole blood without the need for DNA purification, and use of special buffers or complicated protocols. Finnzymes’ solution for High Performance (HP)-PCR results in faster amplification with better yields for raw blood samples as compared with conventional methods for PCR.
Finnzymes’ HP-PCR is a tripartite solution focused on speeding up a PCR and providing marked improvements in yield while maintaining high fidelity. The unique combination of our highly processive PhusionTM DNA Polymerases, quick PikoTM Thermal Cyclers and ultra-thin walled UTWTM tubes provides considerable benefits to the speed, yield, fidelity, specificity and robustness of PCR. Central to these improvements is our Phusion technology, which enhances activity by way of a double-stranded DNA-binding domain bound to a highly engineered proofreading polymerase. This dsDNA-binding domain allows for ultra-robust reactions by tethering the polymerase to the duplex DNA, driving much higher association constants, so the effects of conventional inhibitors of PCR are significantly reduced1. Here we describe one such application, a simple protocol for robust amplification of DNA from whole blood. Whole blood is often used in clinical diagnostics and as a DNA source for PCR tests. Unfortunately, Taq DNA polymerase, the most commonly used enzyme for PCR, is known to be completely inhibited by even small quantities of whole blood in the reaction2. Several strategies have been used to overcome this difficulty, including the use of specialized purifications, buffers and pretreatments3,4, but all of these methods have drawbacks, adding time and cost, and often producing inconsistent results. As a consequence, purification of genomic DNA from whole blood remains a common practice even though it adds considerable time and expense to the test

HP-PCR is resistant to whole blood

Phusion Flash PCR Master Mix has been engineered to work well in the presence of strong PCR inhibitors. We compared Taq DNA polymerase used in a conventional thermal cycler (referred to as conventional PCR) to Phusion Flash PCR Master Mix run in a high-speed Piko Thermal Cycler (referred to as HP-PCR) for the resistance to PCR inhibition in the presence of whole blood. In this case successful PCR requires that genomic DNA is available for amplification and that the polymerase can function in the presence of blood-borne inhibitors. To separate these two factors, we first performed PCR on an exogenous amplicon (λ DNA) in the presence of various quantities of whole blood. We collected human blood and added EDTA as an anticoagulant to a final concentration of 5 mM. Other than the EDTA, we used no other sample preparation or special buffers; whole blood was simply added directly to the PCRs containing 1 pg of purified λ DNA. Cycling was performed according to manufacturer’s recommendations. Conventional PCR (using standard recommended conditions) can amplify a fragment of λ DNA from 1 pg of starting template in the absence of whole blood (Fig. 1a). However, the addition of even 0.2% blood is enough to completely inhibit the conventional PCR. In contrast, HP-PCRs with as much as 1% whole blood showed little or no inhibition, and there is some amplification with even as much as 15% blood. The anticoagulant used in these blood samples was EDTA, which sequesters divalent cations, such as Mg2+. These magnesium ions, which are important for polymerase function, are generally unavailable in reactions with high percentages of whole blood. To overcome this effect we supplemented the buffer with additional MgCl2. Thus,we were able to recover efficient amplification in reactions containing as much as 25% whole blood (Fig. 1b). Using HP-PCR allows the 400–base pair (bp) amplicon to be efficiently produced in less than 20 min, whereas the conventional PCR (which worked only in the absence of whole blood) required almost 60 min.

High-fidelity PCR

We next amplified a 700-bp fragment of a single-copy gene directly from whole blood using HP-PCR. We prepared reactions as described above. To lyse leukocytes, a 5-min incubation at 90 °C was included at the beginning of each protocol. As before, we simultaneously performed conventional PCR according to the manufacturer’s recommended protocol. We ran positive control reactions using 50 ng of purified genomic DNA in the absence of whole blood. Taq-powered conventional PCR amplified the positive control but was completely inhibited by the smallest quantity of whole blood

Conclusion