Pcr hot start pdf

Hot start pcr pdf. Hot start activation approaches are increasingly being used to improve the performance of pcr. Any remaining phusion hot start dna polymerase will degrade the a overhangs, thus creating the blunt ends again. Established approaches to hot start pcr predominately includes three distinct methods: Refer to the troubleshooting section of this protocol for more information about hot start pcr and other alternative methods.

Hotstartaq dna polymerase hotstartaq dna polymerase is a modified form of the recombinant 94 kda taqdna polymerase from qiagen. Suggested cycling parameters for using universe hot start dna polymerase are provided below. By jonathan shum, joyclyn yee, elena hidalgo ashrafi, and natasha paul, ph. Not for use in diagnostic procedures. Kapa hifi hotstart pcr kit technical data sheet 2 for research use only. Hotstartaq dna polymerase is provided in an inactive The same protocol was used for all targets with the annealing and extension settings indicated.

A novel approach for improved pcr performance. If these conditions are not adhered To improve the specificity of pcr, trilink's novel approach to hot start pcr employs chemically modified primers.

The hot start pcr is the most advanced modification of conventional pcr in which one of the pcr reagents is activated only after heating in pcr. This protocol modification avoids likely inactivation of the dna polymerase enzyme. Or pcr from crude extracts, such as those prepared using kapa express extract.

Critical optimization parameters for successful pcr using pfuultra hotstart dna polymerase are outlined in table iii and are discussed in the following section. Accupower Hotstart Pcr Premix. When the program has finished, the 0. PCR products can be detected by loading aliquots of each reaction into wells of an agarose gel then staining DNA that has migrated into the gel following electrophoresis with ethidium bromide.

If a PCR product is present, the ethidium bromide will intercalate between the bases of the DNA strands, allowing bands to be visualized with a UV illuminator. When setting up multiple PCR experiments, it is advantageous to assemble a mixture of reagents common to all reactions i. For instance, if there are 10 x 0. The reagents in the Master Mix are mixed thoroughly by gently pumping the plunger of a micropipettor up and down about 20 times as described above.

Each PCR tube receives an aliquot of the Master Mix to which the DNA template, any required primers, and experiment-specific reagents are then added see Tables 1 and 7. False positives may occur as a consequence of carry-over from another PCR reaction which would be visualized as multiple undesired products on an agarose gel after electrophoresis. Therefore, it is prudent to use proper technique, include a negative control and positive control when possible.

While ethidium bromide is the most common stain for nucleic acids there are several safer and less toxic alternatives. While most modern PCR machines use 0. See your thermal cyclers manual to determine the appropriate size tube.

Knowing the melting temperature T m of the primers is imperative for a successful PCR experiment. Although there are several T m calculators available, it is important to note that these calculations are an estimate of the actual T m due to lack of specific information about a particular reaction and assumptions made in the algorithms for the T m calculators themselves.

The former will give more accurate T m estimation because it takes into account the stacking energy of neighboring base pairs. The latter is used more frequently because the calculations are simple and can be done quickly by hand. See Troubleshooting section for information about how various PCR conditions and additives affect melting temperature.

PCR thermal cyclers rapidly heat and cool the reaction mixture, allowing for heat-induced denaturation of duplex DNA strand separation , annealing of primers to the plus and minus strands of the DNA template, and elongation of the PCR product. Any longer than 3 minutes may inactivate the DNA polymerase, destroying its enzymatic activity. One method, known as hot-start PCR, drastically extends the initial denaturation time from 3 minutes up to 9 minutes.

This protocol modification avoids likely inactivation of the DNA polymerase enzyme. Refer to the Troubleshooting section of this protocol for more information about hot start PCR and other alternative methods. The next step is to set the thermal cycler to initiate the first of 25 to 35 rounds of a three-step temperature cycle Table 2. While increasing the number of cycles above 35 will result in a greater quantity of PCR products, too many rounds often results in the enrichment of undesirable secondary products.

The three temperature steps in a single cycle accomplish three tasks: the first step denatures the template and in later cycles, the amplicons as well , the second step allows optimal annealing of primers, and the third step permits the DNA polymerase to bind to the DNA template and synthesize the PCR product.

The duration and temperature of each step within a cycle may be altered to optimize production of the desired amplicon.

The time for the denaturation step is kept as short as possible. Usually 10 to 60 seconds is sufficient for most DNA templates. The denaturation time and temperature may vary depending on the G-C content of the template DNA, as well as the ramp rate, which is the time it takes the thermal cycler to change from one temperature to the next.

The temperature for this step is usually the same as that used for the initial denaturation phase step 1 above; e. The cycle concludes with an elongation step. The temperature depends on the DNA polymerase selected for the experiment.

Pfu DNA Polymerase is recommended for use in PCR and primer extension reactions that require high fidelity and requires 2 minutes for every 1 kb to be amplified.

See manufacturer recommendations for exact elongation temperatures and elongation time indicated for each specific DNA polymerase. The final phase of thermal cycling incorporates an extended elongation period of 5 minutes or longer.

This last step allows synthesis of many uncompleted amplicons to finish and, in the case of Taq DNA polymerase, permits the addition of an adenine residue to the 3' ends of all PCR products. This modification is mediated by the terminal transferase activity of Taq DNA polymerase and is useful for subsequent molecular cloning procedures that require a 3'-overhang. The stringency of a reaction may be modulated such that the specificity is adjusted by altering variables e.

For example, if the reaction is not stringent enough, many spurious amplicons will be generated with variable lengths. If the reaction is too stringent, no product will be produced. Troubleshooting PCR reactions may be a frustrating endeavor at times.

However, careful analysis and a good understanding of the reagents used in a PCR experiment can reduce the amount of time and trials needed to obtain the desired results. However, before changing anything, be sure that an erroneous result was not due to human error. Start by confirming all reagents were added to a given reaction and that the reagents were not contaminated.

Are there non-specific products bands that migrate at a different size than the desired product? Was there a lack of any product? Also, it is wise to analyze the G-C content of the desired amplicon. First determine if any of the PCR reagents are catastrophic to your reaction. This can be achieved by preparing new reagents e.

This process will determine which reagent was the culprit for the failed PCR experiment. In the case of very old DNA, which often accumulates inhibitors, it has been demonstrated that addition of bovine serum albumin may help alleviate the problem. Primer dimers can form when primers preferentially self anneal or anneal to the other primer in the reaction.

If this occurs, a small product of less than bp will appear on the agarose gel. Start by altering the ratio of template to primer; if the primer concentration is in extreme excess over the template concentration, then the primers will be more likely to anneal to themselves or each other over the DNA template. Adding DMSO and or using a hot start thermal cycling method may resolve the problem.

In the end it may be necessary to design new primers. Non-specific products are produced when PCR stringency is excessively low resulting in non-specific PCR bands with variable lengths.

This produces a ladder effect on an agarose gel. It then is advisable to choose PCR conditions that increase stringency. A smear of various sizes may also result from primers designed to highly repetitive sequences when amplifying genomic DNA.

However, the same primers may amplify a target sequence on a plasmid without encountering the same problem. Lack of PCR products is likely due to reaction conditions that are too stringent.

Primer dimers and hairpin loop structures that form with the primers or in the denatured template DNA may also prevent amplification of PCR products because these molecules may no longer base pair with the desired DNA counterpart. If the G-C content has not been analyzed, it is time to do so.

However, there are many additives that have been used to help alleviate the challenges. Understanding the function of reagents used on conventional PCR is critical when first deciding how best to alter reaction conditions to obtain the desired product. However, the wrong concentration of such reagents may lead to spurious results, decreasing the stringency of the reaction.

When troubleshooting PCR, only one reagent should be manipulated at a time. However, it may be prudent to titrate the manipulated reagent. Changing the magnesium concentration is one of the easiest reagents to manipulate with perhaps the greatest impact on the stringency of PCR.

The 10 X PCR buffer solutions may contain 15 mM MgCl 2 , which is enough for a typical PCR reaction, or it may be added separately at a concentration optimized for a particular reaction. If the desired amplicon is below bp and long non-specific products are forming, specificity may be improved by titrating KCl, increasing the concentration in 10 mM increments up to mM. Thus, choosing an appropriate enzyme can be helpful for obtaining desired amplicon products.

The addition of a 3' adenine has become a useful strategy for cloning PCR products into TA vectors whit 3' thymine overhangs. However, if fidelity is more important an enzyme such as Pfu may be a better choice. Several manufactures have an array of specific DNA polymerases designed for specialized needs. Take a look at the reaction conditions and characteristics of the desired amplicon, and then match the PCR experiment with the appropriate DNA polymerase. Most manufactures have tables that aid DNA polymerase selection by listing characteristics such as fidelity, yield, speed, optimal target lengths, and whether it is useful for G-C rich amplification or hot start PCR.

Optimal target molecules are between 10 4 to 10 7 molecules and may be calculated as was described in the notes above. Additive reagents may yield results when all else fails. Understanding the reagents and what they are used for is critical in determining which reagents may be most effective in the acquisition of the desired PCR product. Adding reagents to the reaction is complicated by the fact that manipulation of one reagent may impact the usable concentration of another reagent.

In addition to the reagents listed below, proprietary commercially available additives are available from many biotechnology companies. Formamide final reaction concentration of 1. Formamide also has been shown to be an enhancer for G-C rich templates. As the amplicon or template DNA is denatured, it will often form secondary structures such as hairpin loops. Betaine final reaction concentration of 0.

Non ionic detergents function to suppress secondary structure formation and help stabilize the DNA polymerase. Non ionic detergents such as Triton X, Tween 20, or NP may be used at reaction concentrations of 0. The presence of non ionic detergents decreases PCR stringency, potentially leading to spurious product formation. However, their use will also neutralize the inhibitory affects of SDS, an occasional contaminant of DNA extraction protocols.

Hot start PCR is a versatile modification in which the initial denaturation time is increased dramatically Table 4. This modification can be incorporated with or without other modifications to cycling conditions.

Moreover, it is often used in conjunction with additives for temperamental amplicon formation. In fact, hot start PCR is increasingly included as a regular aspect of general cycling conditions. Hot start has been demonstrated to increase amplicon yield, while increasing the specificity and fidelity of the reaction.

The rationale behind hot start PCR is to eliminate primer-dimer and non-specific priming that may result as a consequence of setting up the reaction below the T m. In general, the DNA polymerase is withheld from the reaction during the initial, elongated, denaturing time.

Although other components of the reaction are sometimes omitted instead of the DNA polymerase, here we will focus on the DNA polymerase. There are several methods which allow the DNA polymerase to remain inactive or physically separated until the initial denaturation period has completed, including the use of a solid wax barrier, anti-DNA polymerase antibodies, and accessory proteins.

Alternatively, the DNA polymerase may simply be added to the reaction after the initial denaturation cycle is complete. The concept is to design two phases of cycling conditions Table 5. The first phase employs successively lower annealing temperatures every second cycle traditionally 1. The function of the first phase should alleviate mispriming, conferring a 4-fold advantage to the correct product.

Thus, after 10 cycles, a fold advantage would yield copies of the correct product over any spurious priming. This would allow the correct product a fold advantage over false priming products. The concept takes into account a relatively new feature associated with modern thermal cyclers, which allows adjustment of the ramp speed as well as the cooling rate.

The ramp speed is lowered to 2. Nested PCR is a powerful tool used to eliminate spurious products. The use of nested primers is particularly helpful when there are several paralogous genes in a single genome or when there is low copy number of a target sequence within a heterogeneous population of orthologous sequences. The basic procedure involves two sets of primers that amplify a single region of DNA. The outer primers straddle the segment of interest and are used to generate PCR products that are often non-specific in 20 to 30 cycles.

Other PCR protocols are more specialized and go beyond the scope of this paper. The results incorporate several troubleshooting strategies to demonstrate the effect of various reagents and conditions on the reaction. Genes from the budding yeast Saccharomyces cerevisiae and from an uncharacterized Mycobacteriophage were amplified in these experiments. The standard 3-step PCR protocol outlined in Table 2 was employed for all three experiments described below.

The working stocks were prepared as follows. Since the S. This phage DNA is about 67 Kb. Thus, 1 ng contains 2. The working stocks were then used to generate the Master Mix solutions outlined in Table 7. Experiments varied cycling conditions as described below. No MgCl 2 was present in the original PCR buffer and had to be supplemented at the concentrations indicated with a range tested from 0.

The recommended concentration provided by the manufacturer was 1. Perhaps surprisingly, the necessary concentration needed for product formation in this experiment exceeded this amount. A different DNA template was used for the experiment presented in Figure 3b.

As shown in Figure 3b , amplification of the desired PCR product requires at least 2. Notice that in the experiments presented in Figures 3A and 3B , a discrete band was obtained using the cycling conditions thought to be optimal based on primer annealing temperatures. For the third experiment presented in Figure 3c , three changes were made to the cycling conditions used to amplify the yeast GAL3 gene.

Second, the extension time was extended to 1 minute and 30 seconds. Third, the number of cycles was increased from 30 to 35 times. The purpose was to demonstrate the effects of sub-optimal amplification conditions i. As shown in Figure 3c , what was a discrete band in Figure 3a , becomes a smear of non-specific products under these sub-optimal cycling conditions.

These results also demonstrate that when both the cycling conditions are correctly designed and the reagents are at optimal concentrations, the PCR experiment produces a discreet amplicon corresponding to the expected size. The results show the importance of performing PCR experiments at a sufficiently high stringency e. Moreover, the experiments indicate that changing one parameter can influence another parameter, thus affecting the reaction outcome.

The Master Mix depicted in the above table is calculated for 11 reactions plus 2 extra reactions to accommodate pipette transfer loss ensuring there is enough to aliquot to each reaction tube. Table 7.

Figure 1. Note that primers do not always anneal at the extreme ends and may form smaller loop structures. Once the primers anneal to each other they will elongate to the primer ends. Figure 2. Ice bucket with reagents, pipettes, and racks required for a PCR. P pipette, 2. P pipette, 3.