What is polymerase chain reaction?

Polymerase Chain Reaction (PCR) is a molecular biology tool invented by Kary Mullis in the year 1985 to amplify a DNA fragment of interest. PCR utilizes temperature to denature the double helix of DNA. It then anneals the primers in the sequence of interest and thereafter extends the annealed primers by deploying DNA polymerase for generating the intended DNA sequence.

PCR has become an essential technique in nearly every life science or diagnostic field. Applications include genetic engineering, forensic analysis, environmental monitoring, DNA sequencing, diagnosis of disease andgene expression analysis

How does PCR work?

The basic steps in a conventional PCR start with denaturation where the primary goal is to separate the DNA double helix into two separate strands by heating to around 95 °C. This is followed by annealing in which the DNA specimen is cooled to around 50-60 °C to allow the primers to bind the separated DNA strands. The next step, extension, is where the DNA specimen is again subjected to heat that coincides with the polymerase’s optimal temperature. The prime objective of this step is to extend the primers and thereby synthesize the complimentary DNA (cDNA) strands. Lastly, during the amplification phase, the annealing and extension are carried out in multiple rounds to obtain a significant amount of cDNA.

Types of polymerase chain reaction

PCR has been adapted to meet various scientific and medical research needs. A few of the most widely used forms of PCR are detailed below.

Standard PCR

• This type of PCR is also called endpoint or conventional PCR. This is the most widely used form of PCR, which is used to amplify a specific DNA segment.

Reverse transcription PCR (RT-PCR)

• In this type of PCR, the RNA molecule is targeted for amplification. The RNA molecule is reverse transcribed to cDNA using a reverse transcriptase enzyme then amplified through standard PCR.

Quantitative real-time PCR (qPCR or qRT-PCR)

• This form of PCR quantifies the DNA present within the sample by measuring the intensity of a fluorescent probe that adheres to the DNA during amplification. The signal intensity of the target DNA sequence is measured against a standard to determine the relative quantity.

Nested PCR

• This PCR variant encompasses using two primer sets to amplify the DNA fragment of interest. The initial PCR amplifies the greater DNA segment. The subsequent PCR uses the primers nested within the initial PCR product to amplify more specific DNA segments.

Multiplex PCR

• This kind of PCR involves co-amplifying the multiple segments of DNA of interest in a single run using multiple primers. It is a useful tool for detecting more than one pathogen in a sample or for genotyping multiple genetic markers.

Touchdown PCR

• This PCR variant involves the usage of a lowered annealing temperature in every reaction cycle. This type of PCR is designed to elevate the precision of the PCR process through the reduction of non-specific amplification.

Hot start PCR

• This is the modified version of the standard PCR, which includes an initial denaturation step at an elevated temperature to activate the Taq DNA polymerase which in turn brings down non-specific amplification and boosts specificity.

Each type of PCR has its pros and cons and can be used to address various research needs.

What is PCR technique used for?

Gene expression analysis

PCR has a wide spectrum of applications but is frequently used to quantify the existence and/or amount of a specific gene’s expression in various tissues.

Forensic science and DNA analysis

PCR can also boost the amount of a particular DNA sequence for testing or research purposes like pinpointing genetic mutations or sequence variations linked to phenotypic traits or diseases.

Forensic science routinely uses PCR to analyze DNA specimens collected from crime scenes to identify victims or suspects. Archaeology employs PCR to help in the identification of biological specimens that are being collected from historical sites.

Medical diagnosis and pathogen detection

Applied in the field of medical diagnosis for clinical specimens, PCR allows the early sensing and treatment of a spectrum of deadly pathogens and infectious diseases.

Limitations and shortcomings of PCR

False positive results

Although PCR is a very sensitive tool to detect the presence of a very minute quantity of DNA, it can sometimes derive false positive results. A few common causes are presence of contaminating DNA, primers not properly annealing, or poorly optimized amplification conditions.

Cross-reactivity and non-specific amplification

Cross-reactivity with homologous DNA sequences might result in non-specific amplification and interference with the result interpretation. PCR has been reported to amplify DNA sequences unevenly resulting in bias in the representation of different DNA sequences, particularly for samples containing either a low DNA concentration or low-quality DNA.

Length limitations in PCR amplification

The length of the PCR amplified product is limited by the size of the polymerase enzyme used in the reaction which is typically 2-5 kb. Accordingly, PCR may not be practically suitable for amplifying very large DNA fragments. Furthermore, because PCR is a complex and delicate tool, even minute variations in the sample quality or reaction condition might have an impact on the reaction outcome and can be tough to control and standardize.

The PCR user should be aware of these shortcomings and limitations and act accordingly to optimize the reaction parameters, validate the results and use alternate techniques to confirm their findings.

PCR: Troubleshooting tips and techniques

Like all other molecular biology tools and techniques, PCR might sometimes fail and generate suboptimal results due to experimental variables or technical glitches which require some troubleshooting.

Primer design and concentration

Properly designed primers possess specificity to the DNA of interest. It is best to focus on the primer’s concentration and the annealing temperature and adjust accordingly.

DNA quality and quantity

It is also important to ensure availability of inhibitor-free and good quality DNA in ample quantity. Avoid the repetition of the freeze-thaw cycle.

Enzyme and buffer considerations

The quality and concentration of the enzyme DNA polymerase and buffer components should be checked and handled properly. It’s good practice to ensure that the enzyme and the buffer are freshly prepared.

Cycling conditions and annealing temperature

The cycling conditions, annealing temperature, number of cycles and extension time must be carefully optimized.

Reaction volume quantity

It is vital to take the reaction volume in the appropriate quantity as overloading the reaction might result in non-specific or inefficient amplification.

Contamination prevention

Another aspect to look out for is contamination. Use isolated and separate working spaces to avoid the contamination of the reaction. Using dedicated pipettes and protective gear should also be considered as an essential element of a successful PCR experiment.

Alternate techniques for desired results

Moreover, the usage of negative control for monitoring should be ensured. If the standard PCR does not yield the desired results, consider using alternative PCR protocols, such as nested PCR, multiplex PCR, or real-time PCR.

Researchers can increase the likelihood of success and obtain accurate and reliable results by carefully troubleshooting and optimizing the PCR reaction.

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