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Discover the secrets of the number-one used laboratory technique: Polymerase Chain Reaction (PCR)

Discover the secrets of the number-one used laboratory technique: Polymerase Chain Reaction (PCR)

Table of contents

Intro

Polymerase chain reaction (PCR) is one of the most used molecular laboratory techniques.  Different types of PCR are the starting point of many experiments.

In PCR, the target DNA molecule undergoes in vitro amplification, from a few copies to thousands or millions, in three cycling steps, using a heat-stable DNA polymerase enzyme.

Let’s dive in!

Procedure of PCR explained in 3 basic steps

The three cycling steps with predefined temperatures and times that define PCR are:

1. Denaturation step

In the first step of the polymerase chain reaction, the temperature is increased above 90 °C, usually, it is set to 95 °C, for up to 1 minute.

High temperatures break the hydrogen bonds holding together the double-stranded DNA, leading to DNA unwinding and becoming single-stranded.

Single strands of DNA serve as templates for the next PCR step.

2. Annealing step

At the annealing step, the temperature is lowered to the annealing temperature of the designed primers. This is the optimal temperature for the primers to base pair with the complementary region of the template DNA strand.

This step of DNA hybridization is highly temperature-dependent and can be varied, depending on the exact sequence and length of the primers. The usual temperature can be anywhere between 50-60°C, but primers with high GC content can increase the set temperate of the annealing to 72°C.

The annealed primer sequences provide a free 3’ OH end, which serves as a foundation for DNA polymerase activity.

3. Elongation step

The extension or elongation step is the last step of one PCR cycle. At this point thermostable polymerase, most commonly Taq polymerase binds to the free 3’end provided by the annealed primers and starts synthesizing a new DNA strand complementary to the template DNA strand.

The temperature of this cycle is set at 72°C, as this is the optimum of the Taq polymerase.

DNA synthesis occurs at the rate of 1000 bases per minute, therefore PCR extension time for every 500bp of product is set to 30 seconds.

 

Figure 1. The 3 steps of the exponential target DNA amplification using  PCR.

One PCR cycle constitutes of the above described  3 steps. PCR cycle is repeated 25-35 times and after each cycle the newly amplified DNA fragments serve as further templates for the PCR. This process leads to doubled DNA after each cycle thus exponential amplification is achieved. Increasing the number of cycles above 35 will indeed lead to more PCR product, but it can also result in the increase of undesirable secondary products.

PCR thermal cyclers are machines used to perform amplification of DNA though PCR. Thermocyclers rapidly heat and cool down the reaction mixture, allowing for all 3 steps of the cycle to take place.

 

Figure 2. Thermal cycling temperature profile for PCR.

Thermal cyclers have been developed and used for the automation of the PCR, giving the possibility to set the temperatures and times for a particular reaction.

Cycling times depend on the size of the template and the GC content.

The general PCR starts with a one minute denaturation step with a temperature of 94 °C  to 98 °C. Depending on the DNA polymerase optimal activity and the melting temperature of the template DNA. Times longer than 3 minutes may inactivate the enzyme activity of the DNA polymerase.

Next, the first 25-35 rounds of a three-step temperature cycle begin. 

The first step denatures the template and later on DNA amplicons, in 10 -60 seconds. This is followed by a 30-second annealing step, where the temperature is set to about 5 °C below the melting temperature (Tm) of the primers, between 52 °C to 58 °C. The cycle concludes with an elongation step at an optimal temperate for DNA polymerase activity.

In order for the Taq polymerase to finish synthesizing any uncompleted amplicons, there is an extended elongation period of 5 minutes or longer as the final phase of thermal cycling, before the reaction is terminated by chilling the mixture to  4 °C.

Key PCR components

For PCR to work efficiently the mixture should contain the key components, including a DNA template, DNA polymerase enzyme, primers (forward and reverse), nucleotides, and reaction buffer with magnesium ions.

1. DNA Template

    The polymerase chain reaction is a very sensitive method and needs only trace amounts of source DNA in order to amplify it.

    However, optimizing the DNA input in the reaction is still crucial, since low DNA concentrations reduce the amplification of PCR product and high concentrations of DNA can lead to non-specific amplification.

    A rough rule to follow is that the final PCR mixture should contain at least 105 DNA molecules.

    2. Heat stable DNA polymerase (Taq)

      Taq DNA polymerase recognizes a free 3’OH end of a primer. The binding of the polymerase consequently leads to DNA polymerase synthesizing a new DNA strand complementary to the template DNA.

      Taq DNA polymerase is not just any ordinary polymerase, it has a feature that makes it the perfect fit for PCR. It is thermostable! Its temperature optimum is between 75–80°C and it can even survive longer periods on temperatures as high as 96°C.

      However, it also has 2 downsides. First Taq polymerase lacks proofreading activity, meaning it is unable to correct wrongly incorporated bases. The frequency of these errors is quite high and is not desirable, especially for cloning applications. On average, misincorporation occurs once per 9000 nucleotides. Second downside is that DNA fragments to be amplified by Taq polymerase can not be greater than 3kb.

      But do not worry, there is a solution to these problems! There are alternative thermostable polymerases that have proofreading activity, two of them being Pfu and Pwo DNA polymerases.

      3. Primers

        Primers are short, single-stranded nucleic acid sequences used as a starting point for DNA synthesis. Primer sequences provide a free 3’ OH end, which serves as a foundation for DNA polymerase activity.

        In order to multiply the desired target gene region primers need to be complementary to the template region of DNA.

        In PCR 2 types of primers are used, namely forward and reverse primers. They ideally have the same length and do not have sequences complementary to each other.

        5’primers refer to forward primers and 3’primers are reverse primers. This means that the forward primer anneals to the antisense strand of DNA ( 3’ end -> 5’end), whereas reverse primer anneals to the sense strand of DNA ( 5’end -> 3’end). Both primers, forward and reverse,  must have a free 3’ end and they need to be pointed towards each other.

        4. Nucleotides (dNTP)

          Deoxy nucleoside triphosphates (dNTPs) are the four basic nucleotides ( dATP, dCTP, dGTP, and dTTP), which are utilized by the DNA polymerase to synthesize a new DNA strand.

          As a rule, the final concentration of each deoxynucleoside triphosphate in the PCR mixture is 0.2 mM.

          5. Buffer

            Buffer in the PCR reaction mixture provides an aquatic, chemical environment familiar to the DNA polymerase enzyme, which maintains the enzyme activity and stability. The standard pH of the PCR mixture is set to be between 8.0 to 9.5.

            6. Magnesium ions

              In the buffer, an appropriate concentration of magnesium ions (Mg2+), supplied by MgCl2, is crucial for the functioning of the Taq DNA polymerase. Mg2+ ions are a cofactor of the thermostable DNA polymerase and a catalyzer.

              In addition, they stabilize the negative charges on DNA backbones and boost the formation of primer-DNA complexes.

              When Mg2+ concentrations are too low, amplicon production is decreased, due to lower enzyme activity. Whereas excess Mg2+ leads to an increase in the nonspecific product. Thus, standard PCR protocols require a final Mg2+ concentration of 1.5mM.

              Figure 3. Main components of the PCR reaction mixture together with their respective concentrations used in a standard PCR protocol, with a total volume of 50 µl/reaction.

              When preparing to execute PCR it is important to avoid contamination. In PCR design changing one parameter can influence another parameter. That affects the reaction outcome and expected amplicon size. Therefore a positive control should be included if possible. In addition, the negative control should always be included to see if contamination occurred.

              Applications of PCR

              The primary goal of PCR is to exponentially increase the number of target DNA copies in a short amount of time.

              One of the main reasons PCR is a go-to technique is due to the variety of applications. The end PCR product can serve as a sample for further analysis, along with gel electrophoresis and other molecular techniques.

              Amongst the most common applications of PCR are, identifying pathogens, diagnosing infectious diseases, DNA profiling, DNA sequencing, and the list goes on. The wide arrange of applications is summed up in the table below.

              Molecular identification

              Sequencing

              Genetic manipulation

              Detection of pathogens – molecular epidemiology

              Generation of sequencing templates

              Gene expression studies

              Genotyping

              Genomic cloning

              Site-directed mutagenesis

              Mutation screening

              Construction of genetic maps through bioinformatics

              Recombining

              DNA fingerprinting

               

               

              DNA profiling (forensics, paternity tests)

               

               

              Classification of organisms

               

               

              Pre-natal diagnosis

               

               

               

              Conclusion

              As a little treat now that you made it to the end, here is an awesome appreciation song dedicated to PCR by scientists! *Click click*

              https://youtu.be/x5yPkxCLads

              Sources

              • (PDF) Polymerase Chain Reaction (PCR): Back to Basics (researchgate.net)

              • (PDF) Polymerase Chain Reaction (PCR): A Short Review (researchgate.net)

              • Lorenz TC. Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies. Journal of Visualized Experiments : JoVE. 2012;(63):3998. doi:10.3791/3998.