PCR is an enzymatic process in which a specific region of DNA is replicated over and over again to yield many copies of a particular sequence.
The most widely used target nucleic acid amplification method is the polymerase chain reaction (PCR).
This method combines the principles of complementary nucleic acid hybridization with those of nucleic acid replication applied repeatedly through numerous cycles.
This method is able to amplify a single copy of a nucleic acid target, often undetectable by standard hybridization methods, and multiply to 107 or more copies in a relatively short period.
This thus provides ample target that can be readily detected by numerous methods.
Principle of PCR
The target sequence of nucleic acid is denatured to single strands, primers specific for each target strand sequence are added, and DNA polymerase catalyzes the addition of deoxynucleotides to extend and produce new strands complementary to each of the target sequence strands (cycle 1). In cycle 2, both double-stranded products of cycle 1 are denatured and subsequently serve as targets for more primer annealing and extension by DNA polymerase. After 25 to 30 cycles, at least 107 copies of target DNA may be produced by means of this thermal cycling.
Requirements for PCR
- A PCR reaction contains the target double-stranded DNA, two primers that hybridize to flanking sequences on opposing strands of the target, all four deoxyribonucleoside triphosphates and a DNA polymerase along with buffer, co-factors of enzyme and water.
- Since the reaction periodically becomes heated to high temperature, PCR depends upon using a heat-stable DNA polymerase.
- Many such heat-stable enzymes from thermophilic bacteria (bacteria that live in high temperature surroundings) are now available commercially.
- The first one and the most commonly used is the Taq polymerase from the thermophilic bacterium Thermus aquaticus.
Procedure of PCR
A. Extraction and Denaturation of Target Nucleic Acid
- For PCR, nucleic acid is first extracted (released) from the organism or a clinical sample potentially containing the target organism by heat, chemical, or enzymatic methods.
- Once extracted, target nucleic acid is added to the reaction mix containing all the necessary components for PCR (primers, nucleotides, covalent ions, buffer, and enzyme) and placed into a thermal cycler to undergo amplification.
B. Steps in Amplification
- Conventional PCR involves 25 to 50 repetitive cycles, with each cycle comprising three sequential reactions:
- Denaturation of target nucleic acid
- Primer annealing to single-strand target nucleic acid extension of primer target duplex.
- Extension of the primer-target duplex.
- The reaction mixture is heated to 95°C for a short time period (about 15–30 sec) to denature the target DNA into single strands that can act as templates for DNA synthesis.
- The mixture is rapidly cooled to a defined temperature which allows the two primers to bind to the sequences on each of the two strands flanking the target DNA.
- Primers are short, single-stranded sequences of nucleic acid (i.e., oligonucleotides usually 20 to 30 nucleotides long) selected to specifically hybridize (anneal) to a particular nucleic acid target, essentially functioning like probes.
- This annealing temperature is calculated carefully to ensure that the primers bind only to the desired DNA sequences (usually around 55oC).
- One primer binds to each strand. The two parental strands do not re-anneal with each other because the primers are in large excess over parental DNA.
- The temperature of the mixture is raised to 72°C (usually) and kept at this temperature for a pre-set period of time to allow DNA polymerase to elongate each primer by copying the single-stranded templates.
- Annealing of primers to target sequences provides the necessary template format that allows the DNA polymerase to add nucleotides to the 3’ terminus (end) of each primer and extend sequence complementary to the target template
- Taq polymerase is the enzyme commonly used for primer extension, which occurs at 72°C. This enzyme is used because of its ability to function efficiently at elevated temperatures and to withstand the denaturing temperature of 94°C through several cycles.
- The ability to allow primer annealing and extension to occur at elevated temperatures without detriment to the polymerase increases the stringency of the reaction, thus decreasing the chance for amplification of non-target nucleic acid (i.e., nonspecific amplification).
The three steps of the PCR cycle are repeated.
- Thus in the second cycle, the four strands denature, bind primers and are extended. No other reactants need to be added. The three steps are repeated for a third cycle and so on for a set of additional cycles.
- By the third cycle, some of the PCR products represent DNA sequence only between the two primer sites and the sequence does not extend beyond these sites.
- As more and more reaction cycles are carried out, the double-stranded DNA are synthesized more in number. After 20 cycles, the original DNA has been amplified a million-fold and this rises to a billion fold (1000) million after 30 cycles.
C. Product Analysis
- Gel electrophoresis of the amplified product is commonly employed after amplification.
- The amplified DNA is electrophoretically migrated according to their molecular size by performing agarose gel electrophoresis.
- The amplified DNA forms clear bands which can be visualized under ultra-raviolet (UV) light.
Advantages of PCR
- Amplification of DNA: PCR allows for the selective amplification of a specific segment of DNA. This is incredibly useful when working with small or degraded DNA samples, as it can create enough copies for further analysis.
- High Sensitivity: PCR can detect a single or a few copies of a target DNA sequence in a sample. This high sensitivity is crucial in applications like diagnostics, forensics, and research.
- Speed: PCR is a relatively fast technique. The entire process can be completed in a few hours, making it suitable for rapid analysis and diagnostics.
- Versatility: PCR can be adapted for a wide range of applications, such as genotyping, DNA sequencing, DNA cloning, and mutation analysis. It can also be used with different types of DNA, including genomic DNA, cDNA, and RNA.
- Specificity: PCR primers are designed to be highly specific to the target DNA sequence. This specificity ensures that only the desired DNA fragment is amplified, reducing the chance of false-positive results.
- Quantitative PCR (qPCR): PCR can be modified for quantitative analysis, allowing researchers to determine the amount of DNA present in a sample. This is valuable for gene expression studies and quantifying viral or bacterial load.
- Minimal Sample Requirements: PCR can work with very small amounts of DNA, which is particularly advantageous when working with limited or precious samples.
- Automation: PCR can be easily automated, reducing the risk of contamination and human error. High-throughput PCR machines allow for the simultaneous analysis of many samples.
- Conservation of DNA: PCR does not consume the entire DNA sample; only the target region is amplified, leaving the rest of the sample available for future analyses.
- Low Cost: PCR is a relatively cost-effective technique, especially when compared to some alternative methods for DNA analysis.
- Applications in Medicine: PCR has revolutionized diagnostics in medicine, allowing for the detection of genetic diseases, pathogens, and cancer markers. It's also used in prenatal testing and disease monitoring.
- Forensic Applications: PCR is instrumental in forensic science for DNA profiling and matching, helping to solve crimes and establish paternity.
- Research Tool: PCR is a fundamental tool in molecular biology research, facilitating the study of genes, mutations, and DNA sequences.
- Evolutionary Studies: PCR is used in the reconstruction of evolutionary relationships and phylogenetic studies by analyzing DNA sequences from different species.
- Environmental Monitoring: PCR can be employed to detect and identify microorganisms in environmental samples, aiding in pollution assessment and biodiversity studies.
PCR is a versatile and powerful technique with a wide range of applications in various fields, thanks to its speed, specificity, sensitivity, and ability to work with small sample sizes. These advantages have made it an indispensable tool in molecular biology, medicine, genetics, and many other areas of science and research.
Disadventages of PCR
Sensitivity to Contamination: PCR is highly sensitive, which can be an advantage, but it also makes it prone to contamination. Even trace amounts of foreign DNA, including previously amplified PCR products, can lead to false-positive results. Laboratories must employ stringent contamination control measures.
- Limited Quantitative Accuracy: PCR is often used for quantitative purposes, but it may not always provide precise quantitative results. Factors like DNA quality, PCR efficiency, and the presence of inhibitors can affect the accuracy of quantification.
- Amplification Bias: PCR may exhibit amplification bias, meaning it may preferentially amplify certain DNA sequences over others. This bias can be influenced by factors like primer design and template secondary structure, potentially leading to skewed results.
- Limited Size of Amplifiable DNA Fragments: The size of the DNA fragment that can be effectively amplified by PCR is limited. Traditional PCR struggles with very long DNA fragments, and while techniques like long-range PCR exist, they are not as robust.
- Requirement for DNA Template: PCR requires a known DNA template to initiate amplification. This limitation means that it cannot be used for the direct detection of unknown DNA sequences, making it unsuitable for certain applications like metagenomics.
- Labor-Intensive: While technological advancements have streamlined PCR procedures, it can still be labor-intensive and time-consuming, especially when multiple cycles are required for amplification.
- Risk of Cross-Contamination: PCR requires the repeated opening and closing of reaction tubes, creating opportunities for cross-contamination. Precautions must be taken to minimize this risk.
- Cost: PCR can be relatively expensive, particularly when it involves the use of specialized equipment and reagents. High-quality DNA polymerases, primers, and other components can also add to the cost.
- Limited Multiplexing: While multiplex PCR allows the simultaneous amplification of multiple targets, there is a practical limit to the number of targets that can be included in a single reaction due to primer interactions and competition for reagents.
- Evolutionary Analysis Limitations: PCR is limited in its ability to provide information about the evolutionary relationships between different DNA sequences. For this purpose, more advanced techniques like DNA sequencing are often required.
- Risk of Allelic Dropout: In some cases, PCR may fail to amplify specific alleles due to primer-template mismatches, leading to allelic dropout and potentially misleading results.
- Difficulty with GC-Rich or Repeat-Containing Regions: PCR may struggle to amplify DNA regions with high GC content or repetitive sequences due to issues with primer binding and secondary structure formation.
- Limited to DNA: PCR is specific to DNA and cannot be directly used for the amplification of RNA or proteins. Reverse transcription PCR (RT-PCR) is needed to amplify RNA, and protein analysis requires different techniques.
- Limited Resolution: In some cases, PCR products of similar sizes may not be easily distinguishable, limiting its utility in applications where high resolution is required, such as DNA fingerprinting.
PCR remains an essential tool in molecular biology, genetics, forensics, and diagnostics. Researchers have developed various modifications and alternative techniques to address some of these limitations and expand the range of applications.
Applications of PCR
PCR already has very widespread applications, and new uses are being devised on a regular basis.
- PCR can amplify a single DNA molecule from a complex mixture, largely avoiding the need to use DNA cloning to prepare that molecule. Variants of the technique can similarly amplify a specific single RNA molecule from a complex mixture.
- DNA sequencing has been greatly simplified using PCR, and this application is now common.
- By using suitable primers, it is possible to use PCR to create point mutations, deletions and insertions of target DNA which greatly facilitates the analysis of gene expression and function.
- PCR is exquisitely sensitive and can amplify vanishingly small amounts of DNA. Thus, using appropriate primers, very small amounts of specified bacteria and viruses can be detected in tissues, making PCR invaluable for medical diagnosis.
- PCR is now invaluable for characterizing medically important DNA samples. For example, in screening for human genetic diseases, it is rapidly replacing the use of RFLPs.
- Because of its extreme sensitivity, PCR is now fundamentally important to forensic medicine. It is even possible to use PCR to amplify the DNA from a single human hair or a microscopic drop of blood left at the scene of a crime to allow detailed characterization.