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Why would you mix polymerases in PCR?

Why would you mix polymerases in PCR?


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I was reading a manual for kit that requires you to amplify DNA by PCR before analyzing it in the kit. The manual suggests mixing proofreading and non-proofreading polymerases:

For PCR products >2500 bp in length, use a DNA polymerase blend containing Taq DNA polymerase supplemented with a proofreading DNA polymerase

How would this improve the amplification of long products? It suggests using only a proofreading polymerase for fragments below 2500 bases. Does the addition of a non-proofreading polymerase improve speed? I have TAQ and PFU Ultra, but both instructions say to use an extension time of 1 minute per kb.


The Taq polymerase is a rather fast enzyme (the synthesis rate is around 1 kb/min), but the backdraw is that it has no 3'- 5'exonuclease activity which would enable to proofread the newly synthesized DNA. This leads to the introduction of errors and the chance of getting these is higher the longer the DNA you want to synthesize is.

Polymerases which are able to proofread (which have the exonuclease activity) like Pfu have a much lower error rate, but are on the other hand also much slower, typically around 0,5 kb/min. Pfu is also more expensive than the Taq polymerase. See table 1 from reference 1 below:

To combine the advantages of both polymerases, you typically use a blend of Taq and Pfu. Then you get the high processivity of the Taq combined with the proofreading capacity of the Pfu and get good yields of DNA with a low amount of mutations. See also reference 2 for details on this system.

References:

  1. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase
  2. Effective amplification of long targets from cloned inserts and human genomic DNA.

Choose the Right DNA Polymerase for PCR

New England BioLabs has many polymerases designed for use in PCR. How do you decide which one you need? This will depend primarily on your application. The first decision you'll probably make is, do you need high fidelity amplification, or standard amplification? If you plan to use your PCR product in subsequent experiments such as protein expression or sequencing, you want to minimize the incorporation of errors, and you should choose a high-fidelity polymerase. If you just need to see if you have a PCR product or not, for example if you're confirming that your plasmid has an insert, then standard or colony PCR will be appropriate.

For high-fidelity amplification, we recommend starting with Q5 high-fidelity DNA polymerase, since it has the lowest available error rate, and has also been optimized for really robust performance across the ATGC spectrum. For standard PCR or colony PCR, we recommend starting with OneTaq DNA polymerase, which has also been optimized for great performance even with difficult templates such as GC rich.

Next, do you need to set up your reactions at room temperature? Is specificity a challenge? If so, a hot start version of the polymerase will be ideal.

Your next decision will be the polymerase format. For ease of use, we have master mixes that contain the enzyme, buffer, and dNTPs, and also quick load master mix formats that also contain loading dye so that you can load your PCR straight onto the gel. And of course, we also have stand alone polymerases.


DNA Polymerase Proofreading

A 3´→ 5´ proofreading exonuclease domain is intrinsic to most DNA polymerases. It allows the enzyme to check each nucleotide during DNA synthesis and excise mismatched nucleotides in the 3´ to 5´ direction. The proofreading domain also enables a polymerase to remove unpaired 3´ overhanging nucleotides to create blunt ends. Protocols such as high-fidelity PCR, 3´ overhang polishing and high-fidelity second strand synthesis all require the presence of a 3´→ 5´ exonuclease.

In contrast, some applications are enhanced by the use of polymerases without proofreading activity. For example, the efficiency of DNA labeling is enhanced by the absence of proofreading, because it prevents the excision of incorporated bases, allowing for the use of less of the modified base.

Modified base incorporation assays, such as multicolor analysis of gene expression, gene mapping, and in situ hybridization, which utilize DNA that has been labeled with a fluorescent nucleotide to facilitate detection, are well matched to NEB's exonuclease-deficient DNA polymerases. Non-proofreading polymerases are also indispensable when partially filling in 5´ overhangs with only selected dNTPs. Addition of an untemplated dNTP at the 3´ terminus of blunt ends, a requirement for TA cloning, is also promoted by non-proofreading enzymes. In addition to several wild type polymerases in each of these categories, NEB offers genetically altered versions of several proofreading polymerases, where the proofreading exonuclease activity has been attenuated or abolished.


Parameters that affect PCR

Essential components of polymerase chain reactions:

    A thermostable DNA polymerase to catalyse template-dependent synthesis of DNA:

Depending on the ability, fidelity, efficiency to synthesize large DNA products, a wide choice of enzymes is now available. For routine PCRs, Taq polymerase(0.5-2.5 units per standard 25-50 uL reaction)remains the enzyme of choice. The specific activity of most commercial preparations of Taq is

80,000 units/mg of protein. Since the efficiency of primer extension with Taq polymerase is generally

0.7, the enzyme becomes limiting when 1.4*1012 to 7*1012 molecules of amplified product have accumulated in the reaction.

Design of the oligonucleotide primer being the most important factor that influence the efficiency and specificity of the amplification reaction, careful designing of primers is required to obtain the desired products in high yield, to suppress amplification of unwanted sequences and to facilitate subsequent manipulation of the amplified product. Since the primers so heavily influence the success or failure of PCR protocols, it is ironic that the guidelines for their design are largely qualitative and are based more on common sense than on well understood thermodynamic or structural principles.

Design of oligonucleotide primers for PCR

In certain situations, it may be desirable to amplify several segments of target DNA simultaneously. In these cases, an amplification reaction named "multiplex PCR" is used that includes more than one pair of primer in the reaction mix. Standard reactions contain non-limiting amount of primers, typically 0.1-0.5μM of each primer(6*1012 to 3*1013 molecules). This quantity is enough for atleast 30 cycles of amplification of a 1 kb segment of DNA. Higher concentrations of primers favour mispriming which may lead to nonspecific amplification.

Standard PCRs contain equimolar amounts of all four dNTPs. Concentrations of 200-250μM of each dNTP recommended for Taq polymerase in reactions containing1.5 mM MgCl2. In a 50 uL reaction, these amounts should allow synthesis of

6-6.5μg DNA which should be sufficient even for multiplex reaction in which eight or more primer pairs are used at the same time. High concentrations of dNTPs (>4mM) are inhibitory, perhaps because of sequestering of Mg2+. However, a satisfactory amount of amplified product can be produced with dNTP concentrations as low as 20μM- 0.5-1.0pM of an amplified fragment

1 kb in length. To avoid problems, stocks of dNTPs should be stored at -20 o C in small aliquots that should be discarded after the second cycle of freezing or thawing. During long term storage at -20 o C, small amounts of water evaporate and then freeze on the walls of the vial. To minimize changes in concentration, vials containing dNTP solutions should be centrifuged, after thawing, for a few seconds in a micro centrifuge.

All thermostable DNA polymerases require free divalent cations- usually Mg2+ for activity. Some polymerases will also work, albeit less efficiently with buffers containing Mn2+. Calcium ions are quite ineffective. Because dNTPs and oligonucleotides bind Mg2+, the molar concentration of the cation must exceed the molar concentration of phosphate groups contributed by dNTPs and primers. It is therefore impossible to recommend a concentration of Mg2+ that is optional in all circumstances. Although a concentration of 1.5 mM of Mg2+ is routinely used, increasing the concentration of Mg2+ to 4.5 mM or 6mM has been reported to decrease nonspecific priming in some cases and to increase it in others. The optimal concentration of Mg2+ must therefore must be determined empirically for each combination of primers and template. The preparations of template DNA should not contain significant amount of chelating agents (like EDTA or negatively charged ions like PO43-), which can sequester Mg2+.

Tris -Cl ,adjusted to a pH between 8.3 and 8.8 at room temperature is included in standard PCRs at a concentration of 10mM. When incubated at 72 o C(extension phase of PCR), the pH of the reaction mixture drops by more than a full unit, producing a buffer whose pH is

Standard PCR buffer contains 50mM KCl and works well for amplification of segments of DNA >500bp in length. Raising the KCl concentration to

70-100mM often improves the yield of shorter DNA segments.

Template DNA containing target sequences can be added to PCR in single or double stranded form. Closed circular DNA templates are amplified slightly less efficiently than linear DNAs. All though the size of the template DNA is not critical, amplification of sequences embedded in high molecular weight DNA(>10kb) can be improved by digesting the template with a restriction enzyme that doesn't cleave within the target sequence.

When working at its best, PCR requires only a single copy of target sequence as template. More typically, however, several thousand copies of the target DNA are seeded into the reaction. In the case of mammalian genomic DNA,upto 1μg of DNA is utilized per reaction, an amount that contains

3*105 copies of a single-copy autosomal gene. The typical amounts of yeast, bacterial and plasmid DNAs used per reaction are 10μg, 1μg and 1pg respectively.


Choosing a PCR Master Mix

Using a PCR master mix for real-time PCR experiments provides faster setup with less pipetting, reduction in contamination, less tube-to-tube variability, and more reproducible results.

Bio-Rad PCR master mixes are optimized for different applications, for example, gene expression quantification, genotyping, or high-resolution melt analysis. There is a range of considerations when choosing the best PCR master mix for your application including:

  • Type of PCR &mdash screening, cloning, or quantitative
  • Size of the amplicon
  • Template complexity and G-C content, secondary structure, etc.
  • Speed, fidelity, and processivity of the DNA polymerase
  • Inhibitor tolerance
  • Antibody-mediated hot-start polymerase
  • Tolerance to reaction parameters such as different primer Tm values
  • Scalability
  • Cost per reaction

DiyBio Thermocycler Options

There are a few options available to diybio folks who want to run PCR reactions at home. Keeping in mind that all thermocyclers really do is heat and cool DNA samples over a series of cycles, the difference in options comes down to price, size and degree of automation.

Here are some of the options.

MiniPCR mini8

PocketPCR

BentoLab

There was also OpenPCR but it looks like the project has been discontinued and replaced with a qPCR machine that starts at around $5000. I’m going to skip that one since that is out of the range for most diy biologists.

So which thermocycler did I end up choosing?

I weighed all of the options and went in another direction: buying a used thermocycler on eBay!

Buying on eBay is inherently risky, but it can be done. You absolutely need to make sure the listing states the the unit has been tested and works. You absolutely need to make sure it comes with the heat block (the big chunk of metal that the tubes sit in) otherwise, buying a new block can cost more than the machine itself.

I’ve heard some folks get lucky with repairing broken units, but I wouldn’t recommend this approach if you are new to diybio as you’re already going to be overwhelmed with things to do and learn.

I ended up buying an Applied Biosystems GeneAmp PCR 9700 for about $300 ($150 for the unit + $150 for shipping).

This option was the cheapest for me out of all the options mentioned above, even with the high shipping costs. Although it’s not portable and it cannot be controlled via an app, it works. It has a 96-well heat block which is more than I’ll ever need for my home lab. It allows me to easily program and store my PCR routines. And surprisingly, many of the disposables and peripherals are still sold by ThermoFisher.

Top-View of Thermocycler in Shipped Packaging View of the Thermocycler Display

Additional Components Necessary for Running PCR

A thermocycler is only one piece of the puzzle in amplifying DNA. There are other procedures and required tools + reagents you’ll need to successfully run a PCR reaction.

My experience is in extracting and amplifying the ITS gene from fungal samples, but the same general requirements will be needed for any gene(s) you want to amplify:

  • DNA Extraction – you’ll need a sample, protocol for extracting the dna, reagents to break the DNA out of your cultures’ cells, centrifuge, and 1.5ml or .2ml Eppendorf tubes.
  • Oligonucleotides (Primers) primers are little strands of DNA that prefix and suffix the gene(s) you wish to amplify. Once you extract your DNA, you’ll mix in the primers per your protocol prior to running PCR. During PCR, the primers will attach to the gene of interest where the next reagent below will work to double the gene of interest.
  • TAQ Polymerase – this reagent is added with the primers and it’s a special heat-resistant enzyme that facilitates the doubling of the gene(s) of interest between each denaturation / annealing cycle.
  • Buffer – buffer is usually just water and Tris-HCL pH 8.0 (sometimes with EDTA) but it’s role it’s to balance and stabilize the extracted DNA and/or create primer dilutions.

After running PCR, gel electrophoresis can be used to test whether or not your PCR reaction was successful. This is an important step because it ensures you’ve amplified the correct gene before proceeding with costly next steps like sequencing.

In closing, I’ll say that learning PCR is an incredibly useful and versatile tool. Many of the folks I’ve spoken with reflect on their time spent learning and running PCR reactions fondly. Much of modern-day genetic work would not be possible without PCR. So go forth and learn it! It’ll be worth your time.


QPCR and RT-qPCR

Quantitative Endpoint PCR

PCR and RT-PCR are generally used in a qualitative format to evaluate biological samples. However, a wide variety of applications, such as determining viral load, measuring responses to therapeutic agents and characterizing gene expression, would be improved by quantitative determination of target abundance. Theoretically, this should be easy to achieve, given the exponential nature of PCR, because a linear relationship exists between the number of amplification cycles and the logarithm of the number of molecules. In practice, however, amplification efficiency is decreased because of contaminants (inhibitors), competitive reactions, substrate exhaustion, polymerase inactivation and target reannealing. As the number of cycles increases, the amplification efficiency decreases, eventually resulting in a plateau effect.

Normally, quantitative PCR requires that measurements be taken before the plateau phase so that the relationship between the number of cycles and molecules is relatively linear. This point must be determined empirically for different reactions because of the numerous factors that can affect amplification efficiency. Because the measurement is taken prior to the reaction plateau, quantitative PCR uses fewer amplification cycles than basic PCR. This can cause problems in detecting the final product because there is less product to detect.

To monitor amplification efficiency, many applications are designed to include an internal standard in the PCR. One such approach includes a second primer pair that is specific for a &ldquohousekeeping&rdquo gene (i.e., a gene that has constant expression levels among the samples compared) in the reaction (Gaudette and Crain, 1991 Murphy et al. 1990). Amplification of housekeeping genes verifies that the target nucleic acid and reaction components were of acceptable quality but does not account for differences in amplification efficiencies due to differences in product size or primer annealing efficiency between the internal standard and target being quantified.

The concept of competitive PCR&mdasha variation of quantitative PCR&mdashis a response to this limitation. In competitive PCR, a known amount of a control template is added to the reaction. This template is amplified using the same primer pair as the experimental target molecule but yields a distinguishable product (e.g., different size, restriction digest pattern, etc.). The amounts of control and test product are compared after amplification. While these approaches control for the quality of the target nucleic acid, buffer components and primer annealing efficiencies, they have their own limitations (Siebert and Larrick, 1993 McCulloch et al. 1995), including the fact that many depend on final analysis by electrophoresis.

Numerous fluorescent and solid-phase assays exist to measure the amount of amplification product generated in each reaction, but they often fail to discriminate amplified DNA of interest from nonspecific amplification products. Some of these analyses rely on blotting techniques, which introduce another variable due to nucleic acid transfer efficiencies, while other assays were developed to eliminate the need for gel electrophoresis yet provide the requisite specificity. Real-time PCR, which provides the ability to view the results of each amplification cycle, is a popular way of overcoming the need for analysis by electrophoresis.

Quantitative Real-Time PCR

The use of fluorescently labeled oligonucleotide probes or primers or fluorescent DNA-binding dyes to detect and quantitate a PCR product allows quantitative PCR to be performed in real time. Specially designed instruments perform both thermal cycling to amplify the target and fluorescence detection to monitor PCR product accumulation. DNA-binding dyes are easy to use but do not differentiate between specific and nonspecific PCR products and are not conducive to multiplex reactions. Fluorescently labeled nucleic acid probes have the advantage that they react with only specific PCR products, but they can be expensive and difficult to design. Some qPCR technologies employ fluorescently labeled PCR primers instead of probes.

The use of fluorescent DNA-binding dyes is one of the easiest qPCR approaches. The dye is simply added to the reaction, and fluorescence is measured at each PCR cycle. Because fluorescence of these dyes increases dramatically in the presence of double-stranded DNA, DNA synthesis can be monitored as an increase in fluorescent signal. However, preliminary work often must be done to ensure that the PCR conditions yield only specific product. In subsequent reactions, specific amplification can verified by a melt curve analysis. Thermal melt curves are generated by allowing all product to form double-stranded DNA at a lower temperature (approximately 60°C) and slowly ramping the temperature to denaturing levels (approximately 95°C). The product length and sequence affect melting temperature (Tm), so the melt curve is used to characterize amplicon homogeneity. Nonspecific amplification can be identified by broad peaks in the melt curve or peaks with unexpected Tm values. By distinguishing specific and nonspecific amplification products, the melt curve adds a quality control aspect during routine use. The generation of melt curves is not possible with assays that rely on the 5&prime&rarr3&prime exonuclease activity of Taq DNA polymerase, such as the probe-based TaqMan® technology.

Some qPCR strategies employ complementary nucleic acid probes to quantify the DNA target. These probes also can be used to detect single nucleotide polymorphisms (Lee et al. 1993 Bernard et al. 1998). There are several general categories of real-time PCR probes, including hydrolysis, hairpin and simple hybridization probes. These probes contain a complementary sequence that allows the probe to anneal to the accumulating PCR product, but probes can differ in the number and location of the fluorescent reporters.

Hydrolysis probes are labeled with a fluor at the 5&prime-end and a quencher at the 3&prime-end, and because the two reporters are in close proximity, the fluorescent signal is quenched. During the annealing step, the probe hybridizes to the PCR product generated in previous amplification cycles. The resulting probe:target hybrid is a substrate for the 5&prime&rarr3&prime exonuclease activity of the DNA polymerase, which degrades the annealed probe and liberates the fluor (Holland et al. 1991). The fluor is freed from the effects of the energy-absorbing quencher, and the progress of the reaction and accumulation of PCR product is monitored by the resulting increase in fluorescence. With this approach, preliminary experiments must be performed prior to the quantitation experiments to show that the signal generated is proportional to the amount of the desired PCR product and that nonspecific amplification does not occur.

Hairpin probes, also known as molecular beacons, contain inverted repeats separated by a sequence complementary to the target DNA. The repeats anneal to form a hairpin structure, where the fluor at the 5&prime-end and a quencher at the 3&prime-end are in close proximity, resulting in little fluorescent signal. The hairpin probe is designed so that the probe binds preferentially to the target DNA rather than retains the hairpin structure. As the reaction progresses, increasing amounts of the probe anneal to the accumulating PCR product, and as a result, the fluor and quencher become physically separated. The fluor is no longer quenched, and the level of fluorescence increases. One advantage of this technique is that hairpin probes are less likely to mismatch than hydrolysis probes (Tyagi et al. 1998). However, preliminary experiments must be performed to show that the signal is specific for the desired PCR product and that nonspecific amplification does not occur.

The use of simple hybridization probes involves two labeled probes or, alternatively, one labeled probe and a labeled PCR primer. In the first approach, the energy emitted by the fluor on one probe is absorbed by a fluor on the second probe, which hybridizes nearby. In the second approach, the emitted energy is absorbed by a second fluor that is incorporated into the PCR product as part of the primer. Both of these approaches result in increased fluorescence of the energy acceptor and decreased fluorescence of the energy donor. The use of hybridization probes can be simplified even further so that only one labeled probe is required. In this approach, quenching of the fluor by deoxyguanosine is used to bring about a change in fluorescence (Crockett and Wittwer, 2001 Kurata et al. 2001). The labeled probe anneals so that the fluor is in close proximity to G residues within the target sequence, and as probe annealing increases, fluorescence decreases due to deoxyguanosine quenching. With this approach, the location of probe is limited because the probe must hybridize so that the fluorescent dye is very near a G residue. The advantage of simple hybridization probes is their ability to be multiplexed more easily than hydrolysis and hairpin probes through the use of differently colored fluors and probes with different melting temperatures (reviewed in Wittwer et al. 2001).

Some qPCR strategies employ complementary nucleic acid probes to quantify the DNA target. These probes also can be used to detect single nucleotide polymorphisms (Lee et al . 1993 Bernard et al . 1998). There are several general categories of real-time PCR probes, including hydrolysis, hairpin and simple hybridization probes. These probes contain a complementary sequence that allows the probe to anneal to the accumulating PCR product, but probes can differ in the number and location of the fluorescent reporters.

QPCR Products and Resources

GoTaq® qPCR and RT-qPCR kits are available for dye-based or probe-based real-time PCR approaches. GoTaq qPCR Systems contain BRYT Green Dye, which provides maximum amplification efficiency and greater fluorescence than SYBR Green.

GoTaq® Probe Systems are ready-to-use master mixes that simplify reaction assembly for hydrolysis probe-based detection.


In vitro Mutagenesis

PCR Mutagenesis of Circular DNA

PCR mutagenesis can also be used to amplify the entire plasmid containing the gene of interest. One straightforward method, termed ‘inverted’ or ‘counter’ PCR, uses back-to-back primers ( Figure 2B ) one PCR primer serves as the mutagenic oligonucleotide and the other oligonucleotide primes from the opposite strand, adjacent to the mutagenic primer. The PCR product is a full-length, linear plasmid which is then phosphorylated and ligated before transformation. This method can readily be used to make deletion mutants by creating a gap between the primers. Variants of this method include ‘recombinant circle’ PCR ( Figure 2C ) and ‘recombination’ PCR, both of which rely on recombination of linear plasmids. In these techniques, two inverse PCRs are performed with gapped primers at different sites. These two mutant plasmids are then recombined in vitro by mixing and annealing (recombinant circle PCR) or in vivo (recombination PCR). The gaps are then repaired by the bacterial DNA repair machinery.


Advantage HD Polymerase Mix

The Advantage HD Polymerase Mix provides maximum fidelity and extended PCR product length, making it particularly useful for applications that require high accuracy, such as cDNA cloning, site-directed mutagenesis, and mutation genotyping (i.e., SNP analysis).

The Advantage HD Polymerase Mix consists of a novel DNA polymerase with a hot-start antibody, and comes with a tube of 5X buffer (with Mg 2+ ).

The Advantage HD Polymerase Mix provides maximum fidelity and extended PCR product length, making it particularly useful for applications that require high accuracy, such as cDNA cloning, site-directed mutagenesis, and mutation genotyping (i.e., SNP analysis).

The Advantage HD Polymerase Mix consists of a novel DNA polymerase with a hot-start antibody, and comes with a tube of 5X buffer (with Mg 2+ ).

Advantage HD Polymerase Mix provides outstanding accuracy due to the presence of 3' &rarr 5' exonuclease activity that results in an extremely low error rate. It also performs extremely well on GC-rich and other more complex templates. The Advantage HD Polymerase Mix amplifies targets of up to 8.5 kb from human genomic DNA, 10 kb from E. coli genomic DNA, and 22 kb from lambda DNA. The amplification efficiency of Advantage HD Polymerase Mix is greater than that of wild-type Taq polymerase.


PrimeSTAR Max DNA Polymerase—fast and high-fidelity PCR

PrimeSTAR Max DNA Polymerase has the highest fidelity and fastest extension rate of any Takara Bio enzyme. The formulation is based on Takara's unique, high-fidelity PrimeSTAR HS DNA Polymerase combined with an elongation factor to provide efficient priming and extension, which greatly reduces the time required for annealing and extension steps. As a result, PrimeSTAR Max DNA Polymerase can be used for exceptionally fast PCR. It is formulated as a 2X premix containing optimized buffer and dNTPs, which allows for quick preparation of reactions for high-throughput applications.

PrimeSTAR Max DNA Polymerase has the highest fidelity and fastest extension rate of any Takara Bio enzyme. The formulation is based on Takara's unique, high-fidelity PrimeSTAR HS DNA Polymerase combined with an elongation factor to provide efficient priming and extension, which greatly reduces the time required for annealing and extension steps. As a result, PrimeSTAR Max DNA Polymerase can be used for exceptionally fast PCR. It is formulated as a 2X premix containing optimized buffer and dNTPs, which allows for quick preparation of reactions for high-throughput applications.

In addition, standardization of the extension step time makes PrimeSTAR Max DNA Polymerase suitable for reactions with excessive nucleic acids that would ordinarily be difficult to amplify due to non-specific binding. The superior processivity of PrimeSTAR Max DNA Polymerase prevents inhibition by excessive nucleic acids, resulting in a much higher success rate for PCR with minimal required optimization. The antibody-mediated hot-start feature provides improved specificity by preventing false initiation events during the reaction assembly.


Watch the video: Hotel quarantine and RT PCR test (June 2022).


Comments:

  1. Doucage

    So cute))

  2. Kearn

    I must tell you this is a false path.

  3. Sterne

    In it something is. I thank for the information.

  4. Tulkree

    Authoritative answer



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