Polymerase Chain Reaction-Based Techniques for the Diagnosis of Genetic Disorders

Polymerase chain reaction (PCR) is a molecular biology technique that can exponentially amplify a specific DNA fragment in vitro. The PCR was invented by Kary Mullis in 1983 and received the Nobel Prize in Chemistry in 1989. PCR technique has a wide range of applications in life sciences, medicine, forensics, environmental sciences, etc., such as gene cloning, gene expression analysis, genetic disease diagnosis, pathogen detection, paternity testing, ancient DNA analysis, etc.

Fig.1 Schematic presentation of the PCR principle. (Garibyan L, 2013)

The advantages of the PCR technique are high sensitivity, high specificity, simple operation, and fast results. However, the PCR technique also has some limitations, such as amplification efficiency being affected by various factors, amplification products being prone to contamination, amplification bias leading to distorted results, etc. To overcome these limitations or to meet different experimental purposes, people have developed many different types and variants of PCR, such as reverse transcriptase PCR (RT-PCR), real-time PCR (qPCR), allele-specific amplification (ASA), amplification-refractory mutation system (ARMS) PCR, multiplex PCR (mPCR), digital PCR (dPCR), etc. These PCR techniques differ in principle, steps, applications, advantages, and disadvantages and need to be selected according to specific situations.

Reverse Transcriptase PCR

Reverse transcriptase PCR (RT-PCR) is a technique that uses reverse transcriptase to convert RNA into cDNA and then uses PCR to amplify cDNA. RT-PCR steps include RNA extraction, reverse transcription reaction, PCR amplification, and electrophoresis detection. RT-PCR applications include detecting RNA viruses, expression profile analysis, gene cloning, etc. RT-PCR's advantages are high sensitivity, high specificity, and simple operation. RT-PCR limitations include complex operation, proneness to contamination, amplification efficiency affected by various factors, etc.

Real-Time PCR

Real-time PCR (qPCR) is a technique that monitors the amount of amplification products in real time during the PCR process. qPCR steps include DNA extraction, primer design, qPCR reaction, and data analysis. qPCR applications include quantitative analysis, gene genotyping, mutation detection, etc. qPCR's advantages are high accuracy, high efficiency, and high sensitivity. qPCR limitations include high cost, standard curve requirements, primer interference, etc.

Fig.2 Real-time PCR detection chemistries. (Morteza Seifi, 2012)

Allele-Specific Amplification (ASA) By Amplification Refractory Mutation System (ARMS) PCR

Allele-specific amplification (ASA) by amplification refractory mutation system (ARMS) PCR is a technique that uses specific primers to amplify specific alleles. ARMS PCR steps include DNA extraction, primer design, ARMS PCR reaction, and electrophoresis detection. ARMS PCR applications include single nucleotide polymorphism (SNP) analysis, genetic disease diagnosis, etc. ARMS PCR advantages are simplicity and speed, high specificity, and no need for post-processing. ARMS PCR limitations include false-positives possible, critical primer design, amplification efficiency affected by various factors, etc.

Multiplex PCR

Multiplex PCR (mPCR) is a technique that uses multiple pairs of primers to simultaneously amplify multiple target sequences in the same reaction system. mPCR steps include DNA extraction, primer design, mPCR reaction, and electrophoresis detection. mPCR applications include multiplex detection, microbial identification, forensics, etc. mPCR advantages include saving time and materials, increasing throughput, and high sensitivity. mPCR limitations include primer interference, uneven amplification efficiency, difficult optimization, etc.

Digital PCR

Digital PCR (dPCR) is a technique that divides the PCR reaction system into many small-volume compartments and then amplifies and detects each compartment, thereby directly obtaining the copy number of the sample. dPCR steps include DNA extraction, primer design, dPCR reaction, and data analysis. dPCR applications include low-abundance target molecule detection, copy number variation (CNV) analysis, gene editing detection, etc. dPCR advantages are high sensitivity, high accuracy, no need for a standard curve, and anti-interference. dPCR limitations include expensive equipment, complex operation, low throughput, etc.

Nested PCR

Building upon the principles of traditional PCR, nested PCR involves two successive rounds of amplification using two sets of primers. The first round utilizes outer primers to amplify the target DNA region, followed by a second round using inner primers nested within the first amplicon. This nested approach increases the selectivity of the reaction, as the inner primers target a smaller and more specific region, reducing the likelihood of non-specific amplification. Nested PCR is particularly valuable in situations where the initial template concentration is low or when a high degree of specificity is crucial for accurate detection of target sequences.

Conclusion

The PCR technique is a powerful and flexible molecular biology technique that can be adjusted and optimized according to different experimental purposes and conditions to improve sensitivity, specificity, accuracy, and efficiency. The PCR technique has a wide range of applications in life sciences, medicine, forensics, environmental sciences, etc., especially in genetic disease diagnosis and gene therapy. The PCR technique can provide rapid, accurate, and convenient detection and intervention methods, which offer the possibility of improving the quality of life and prognosis of patients. However, the PCR technique also has some limitations and challenges, such as amplification efficiency being affected by various factors, amplification products being prone to contamination, amplification bias leading to distorted results, etc. Therefore, future research needs to further improve the reliability and stability of PCR techniques as well as develop more novel PCR techniques to meet the growing demand for detection. In addition, future research also needs to explore the combination and synergy of the PCR technique with other molecular biology techniques to increase the complexity and diversity of detection. In summary, the PCR technique is a molecular biology technique with great potential and prospects that will continue to make important contributions to scientific progress and social development.

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