With so many PCR enzymes available, choosing the right one can be challenging. The various enzymes used to amplify DNA differ in their accuracy, speed, and specificity. The following three questions can help you sort out which factors to focus on when selecting your PCR enzymes.
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Sometimes you only need to detect a PCR product or estimate its size, such as, when you’re genotyping mice or screening for recombinant clones. For this type of routine PCR, you should use a standard thermostable DNA polymerase (like Taq DNA polymerase) to confirm the presence or absence of target DNA.
If, however, you are performing cloning experiments or next-generation sequencing (NGS), then accuracy matters. To obtain an exact DNA copy, make sure that you are choosing a DNA polymerase that has high-fidelity. High fidelity DNA polymerases have the ability to proofread the DNA sequence being amplified through 3' to 5' exonuclease activity. When a mismatched base pair is incorporated, the DNA polymerase stalls which creates a delay in synthesis. This delay allows the mismatched nucleotide to be removed and replaced with the correct nucleotide.
An excellent choice for a DNA polymerase that preserves sequence accuracy is Thermo Scientific Phusion Plus DNA Polymerase. This enzyme features high fidelity that is >100x more accurate than Taq DNA polymerase. For even higher accuracy of sequences, Invitrogen Platinum SuperFi II DNA Polymerase is available and offers >300x than Taq DNA polymerase's fidelity.
When you’re working with long DNA templates, you need a PCR enzyme that can go the distance. In this case, choose a polymerase with a high processivity and fast extension rate. Processivity represents the number of nucleotides that can be incorporated during a single binding event by the DNA polymerase. Highly processive DNA polymerases help with amplification of long templates due to the number of nucleotides that can be processed. In addition, DNA polymerases with fast extension rates are able to amplify DNA in a shorter time. PCR enzymes with high processivity and fast extension rates will help ensure efficient DNA synthesis of long templates and cut down on your cycling time. Both Platinum SuperFi II and Phusion DNA polymerases can synthesize up to 20 kb of DNA at 15–30 sec/kb.
Maybe you are seeing bands on your gel that should not be there. These extra bands could be an example of nonspecific amplification (see Figure 1). To help achieve specific target amplification with high yields, choose a hot-start PCR enzyme like Platinum II Taq or DreamTaq Hot-Start DNA polymerases. Hot-start DNA polymerases begin amplification only when the initial denaturation step reaches 90°C. This feature helps ensure that the PCR reaction does not initiate too soon, preventing undesirable off-target products. This feature also helps prevent primer-dimers from extending, which is particularly helpful when using multiple primers for multiplex and high-throughput PCR.
Figure 1. Nonspecific amplification (left) vs specific amplification using a hot-start DNA polymerase (right).
There’s more to consider. Selecting the right polymerase format can simplify your workflow. PCR enzyme format options include ready-to-use master mixes, buffers with dyes for direct gel loading, and kits that assemble all the necessary components for direct PCR.
With a master mix, you can just add your template and primers, then start your PCR. If you want to further minimize pipetting steps, use a polymerase with a buffer and loading dye that allows for direct gel loading of PCR products (note: ensure the dye is compatible with downstream applications). Direct PCR kits can also help save time by allowing you to skip purification of DNA and go straight to DNA amplification. Despite these available options, if you’re looking to optimize specific components of your reaction, stick with a stand-alone DNA polymerase.
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As you evaluate the level of accuracy, speed, and specificity you need for your PCR experiments, you can confidently navigate the crowded marketplace of PCR enzymes and choose the right DNA polymerase for your molecular biology applications.
Direct PCR is a technique that allows scientists to go directly from plant, tissue, or cell culture samples to PCR without DNA isolation or purification steps. The elimination of these laborious extra steps provides tremendous time and resource savings for improved sustainability. Our Extract-N-Amp™ PCR kits are unique as they deliver a combined extraction and amplification process for plant, tissue, and blood sample assays. By incorporating a “lyse & go” method, Extract-N-Amp™ PCR removes the requirement for columns or long enzymatic sample purification. Our direct PCR kits come with the reagents, enzymes, and proprietary buffers that are required to quickly extract and amplify targets of interest from a variety of plant, blood, and tissue samples.
Hot Start PCR is a technique that inhibits Hot Start Taq polymerase activity or the incorporation of modified dNTPs during reaction set up until a heat activation step occurs. Hot Start PCR allows for reaction set up at room temperature without non-specific amplification and primer dimer formation. Discover our wide selection of Hot Start PCR enzymes including KOD, FastStart™, JumpStart™, and KAPA Biosystems reagents. For additional information on Hot Start PCR reagents and product information, please navigate the product selection table to meet your Hot Start PCR needs.
KOD DNA Polymerase is an ultra-high-fidelity, thermostable DNA polymerase. Numerous independent studies have also verified the superior high-fidelity of KOD DNA Polymerase compared to other thermophilic polymerases. In addition to a low mutation frequency, the fast extension rate and high processivity of KOD polymerase results in higher yields of full-length product in fewer reaction cycles. Combined, these make KOD DNA polymerases the PCR enzyme of choice when speed and fidelity matter. Explore our new KOD One™ PCR Master Mix, a ready-to-use 2x PCR master mix containing a novel, genetically modified KOD DNA polymerase (UKOD), along with a new elongation accelerator, enabling fast PCR with an extension time of 5 sec/kb for template DNA <10kb.
In order to advance your molecular biology research, it is critical to maximize the efficiency of your workflow in a multitude of areas including, genomics, proteomics, and cellular analyses. As a pioneer in genomics and PCR technology, Roche’s complete suite of reagents and kits are optimized to reduce hands-on time at the bench, expedite data acquisition and the creation of key tools to test the most challenging of biological hypotheses. With a mutual drive for product quality and scientific excellence, our partnership allows us to bring Roche products to you, so the answers are there when you need them, wherever your work takes you. Explore all Roche PCR-related reagents and protocols on the Roche Products and Molecular Solutions resource page.
Quantitative PCR (qPCR) uses the linearity of DNA amplification to determine absolute or relative amounts of a known sequence in a sample. By using a fluorescent reporter in the reaction, it is possible to measure DNA generation in real time. Based on a highly optimized ReadyMix™ chemistry, LuminoCt™ delivers unsurpassed assay speeds without sacrificing accuracy, precision, or sensitivity. LuminoCt™ works seamlessly with most qPCR instruments, so optimization of reaction conditions is kept to an absolute minimum. Often, users can simply choose their kit, build their assay, and begin. Generating the best possible qPCR data has never been faster or easier.
KiCqStart® ReadyMix™ reagents are ready-to-use, highly sensitive master mixes containing all the basic components for qPCR or RT-qPCR. Simply add your primers, probe and water to complete the assay cocktail. Add the cocktail to tubes or plates and then add your template. The KiCqStart® ReadyMix™ reagents are compatible with conventional or fast PCR mode. For more details, see product usage information at KiCqStart® Probe qPCR ReadyMix™ or KiCqStart® One-Step Probe RT-qPCR ReadyMix™ reagents. Additionally, the JumpStart Taq ReadyMixes provide a convenient solution for conventional qPCR experiments. The JumpStart Taq antibody delivers antibody-inactivated hot-start PCR which prevents non-specific product formation. Suitable for SYBR® Green and probe-based detection methods, the product line features options for optimization and offer compatibility with tube-, plate-, and capillary-based instruments. For additional information, please explore our additional SYBR® Green based qPCR reagents and resources.
RT-PCR combines two powerful and versatile techniques, reverse transcription and the polymerase chain reaction to generate and amplify cDNA from total RNA or mRNA transcripts. The method is often used to study gene expression at both the RNA and protein levels. The ideal RT-PCR requires a sensitive reverse transcription and a high-fidelity amplification. The reverse transcriptase should be able to detect very low abundance transcripts and/or transcripts containing difficult secondary structure. Our enhanced Avian (eAMV™) Reverse Transcriptase displays all of these characteristics. It is the ideal RT for detecting low abundance messages that may be missed by other reverse transcriptases and it is the best enzyme we have found for transcribing through difficult secondary structure. It is also more tolerant to elevated temperatures than standard AMV, M-MLV, M-MLV RNase H– or AMV RNase H reduced.
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