Introduction
Standard PCR using Taq DNA polymerase is a powerful technique used in genetic analysis for a variety of applications. For some researchers, the ability to amplify longer targets opens up more possibilities to advance their research
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,
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,
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. However, standard PCR using Taq DNA polymerase is limited to amplifying targets up to 3–4kb in length. This limitation of Taq DNA polymerase is due to its relatively low fidelity and inability to correct nucleotide misincorporations during strand synthesis. Misincorporated nucleotides have a higher extension KM value. This is thought to cause the polymerase to dissociate from the DNA strand, terminating extension and leading to a truncated product
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,
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. These truncated products cannot be used as a template in subsequent amplification cycles, and consequently little or no PCR fragments of the correct length are produced.
Proofreading DNA polymerases such as Pfu, Vent® and Deep Vent® polymerase have higher fidelity than Taq DNA polymerase due to their 3’→5’ exonuclease (i.e., proofreading) activity. This exonuclease activity increases the fidelity of the enzyme by repairing misincorporated nucleotides
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. However, the strong exonuclease activity competes with the polymerization activity and leads to poor amplification efficiency. The poor amplification efficiency makes these proofreading DNA polymerases unable to amplify PCR targets longer than about 2–3kb
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,
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,
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. The proofreading activity reduces the DNA polymerase's efficiency and is considered the cost of proofreading.
The key to amplifying targets longer than 3–4kb is a mixture of two DNA polymerases
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,
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. When a high concentration of Taq or a Taq-like DNA polymerase is blended with a small concentration of a proofreading DNA polymerase, both enzymes work together to amplify long targets with high yield. Taq DNA polymerase does most of the strand synthesis but when it misincorporates a nucleotide, the proofreading polymerase uses its 3’→5’ exonuclease activity to remove the incorrect nucleotide, which allows strand synthesis to continue (Figure 1).
Including proofreading DNA polymerase in a reaction mixture increases the fidelity of the PCR reaction by correcting the errors of the Taq DNA polymerase. The error rate of these Taq DNA polymerase blends has been shown in PCR-based forward mutation assays and 454 pyrosequencing to be higher than proofreading DNA polymerases alone. However, the error rate of these blends is lower than that of Taq DNA polymerase alone, thus giving long PCR DNA polymerase mixtures an intermediate fidelity
(4)
,
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. The intermediate fidelity of long DNA polymerase mixtures allows for amplification of human genomic targets up to 30kb in length and amplification of even longer targets may be possible with less complex DNA templates. Amplification of very long targets is not possible using either Taq or proofreading DNA polymerases alone
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,
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,
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.
The GoTaq® Long PCR Master Mix is a ready-to-use solution for amplifying long PCR targets. The master mix has been formulated with an optimized buffer, dNTPs, MgCl2 and a hot-start enzyme blend for long PCR. The GoTaq® Long PCR Master Mix uses a blend of GoTaq® Hot Start Polymerase and a recombinant proofreading DNA polymerase. GoTaq® Hot Start Polymerase is a hot-start, recombinant Taq DNA polymerase bound by proprietary antibodies that inhibit polymerase activity at lower temperatures. This allows reactions to be set up at room temperature. The polymerase activity is restored by an initial denaturation cycle at high temperature
(8)
. In addition to GoTaq® Hot Start Polymerase, the GoTaq® Long PCR Master Mix contains a small amount of a DNA polymerase with 3’→5’ exonuclease (i.e., proofreading) activity to repair misincorporations from Taq DNA polymerase. Together, the two DNA polymerases can amplify long targets with high yield (Figure 2).
PCR Performance
The GoTaq® Long PCR Master Mix has been formulated to amplify PCR targets 5kb and larger. Smaller targets can be amplified; however, the master mix was designed for longer amplicons. The GoTaq® Long PCR Master Mix can amplify targets up to 30kb in length from human genomic DNA (Figure 3) and up to 40kb in length from lower complexity targets such as lambda DNA (Figure 4). The reactions shown here were set up at room temperature with no reagent optimization.
The sensitivity and high yield of the GoTaq® Long PCR Master Mix can be seen in the amplification of a 17.5kb β-globin target from human genomic DNA (Figure 5). Figure 5 shows that input DNA in amounts smaller than the recommended 0.1–0.5µg for human genomic targets can produce a high yield of product. The 17.5kb β-globin target was amplified from as little as 2ng of human genomic DNA; however, when targets are over 20kb in length, a higher amount of input template may be required (data not shown).
Figure 5. Amplification of a 17.5kb fragment from the human β-globin gene cluster using GoTaq® Long PCR Master Mix. The high yield and sensitivity of GoTaq® Long PCR Master Mix is demonstrated by the successful amplification of the 17.5kb target in reactions containing 200ng (lane 1), 20ng (lane 2), and 2ng (lane 3) of human genomic DNA. Lane M, Lambda DNA/HindIII Markers (Cat.# G1711).
Hot-Start Performance
The GoTaq® Long PCR Master Mix uses GoTaq® Hot Start Polymerase as its primary DNA polymerase. This allows reactions to be assembled at room temperature in addition to providing the flexibility of leaving the assembled reactions at room temperature before cycling. No difference in performance has been observed between amplification reactions that were set up at room temperature and then cycled immediately and reactions that were incubated on the bench for 6 hours before cycling (Figure 6).
Figure 6. Performance with GoTaq® Long PCR Master Mix is not affected by incubation of assembled reactions at room temperature for 6 hours before cycling. Amplification reactions set up at room temperature and then cycled immediately (lane 1) were compared to reactions that incubated at room temperature for 6 hours before cycling (lane 2). Lane M, Lambda DNA/HindIII Markers (Cat.# G1711).
Competitor Analysis
When amplifying a 17.5kb β-globin target sequence, the GoTaq® Long PCR Master Mix performed as well as or better than other long PCR reagents (Figure 7). In addition, the master mix design and hot-start format results in simpler reaction setup than other leading long PCR reagents on the market.
Characteristics and Applications
GoTaq® Hot Start Polymerase, which is the primary DNA polymerase used in the GoTaq® Long PCR Master Mix, has similar characteristics to Taq DNA polymerase. It has 5´→3´ exonuclease activity and an extension rate of 1 minute per 1kb of target DNA
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. The 3´→5´ exonuclease activity in the master mix comes from the proofreading DNA polymerase also present in the mix. The majority of PCR products amplified using the GoTaq® Long PCR Master Mix will have a single deoxyadenosine at the 3´ end. Some blunt-ended fragments will be present due to the small concentration of the proofreading DNA polymerase; however, the terminal transferase activity of the GoTaq® Hot Start Polymerase, which is the predominant enzyme component in the blend, adds a single A to the 3´ end of most PCR fragments. Following purification of the PCR product, these PCR fragments can be used in T-vector cloning applications.
Conclusion
The GoTaq® Long PCR Master Mix is an easy-to-use, reagent for long PCR. The combined hot-start and master-mix format allows reactions to be set up at room temperature with little to no reagent optimization and offers the flexibility of leaving the reactions at room temperature for up to 6 hours before cycling. The GoTaq® Long PCR Master Mix contains everything but primers, template and water to successfully amplify targets up to 30kb in length from human genomic DNA and up to 40kb in length from less complex templates such as lambda DNA. The GoTaq® Long PCR Master Mix requires little optimization and performs as well as or better than competing long PCR reagents.