ABSTRACT:
In this article, we will discuss about the types polymerase chain reaction. It is a laboratory technique use to amplify and create multiple copies of specific segment of DNA/RNA. There are several types of PCR: inverse PCR, reverse transcriptase PCR, multiplex PCR, etc. We have to move towards the different types PCR due to some demerits of traditional PCR such as non-specific binding and amplification, primer-dimer formation and various other deficiencies. Here, we will discuss about the principles and procedure of different types of PCR. We will also provide references to learn and acknowledge the concept of PCR.
INTRODUCTION-TYPES OF POLYMERASE CHAIN REACTION:
Polymerase Chain Reaction (PCR) is a revolutionary molecular biology technique that amplifies DNA sequences, allowing for their analysis and manipulation. Since its invention by Kary Mullis in 1983, PCR has evolved, leading to various specialized types tailored to specific applications. In this article, we’ll delve into some prominent types of PCR and their applications.
TYPES OF POLYMERASE CHAIN REACTION:
1. CONVENTIONAL PCR:
Conventional PCR is the foundational method that involves three main steps – denaturation, annealing, and extension. It amplifies a specific DNA region and widely used in diagnostics, research, and forensics. (Mullis, et al., 1986)
2. REVERSE TRANSCRIPTION PCR-MOST ABUNDANT TYPE OF POLYMERASE CHAIN REACTION:
RT-PCR employed to amplify RNA rather than DNA. It begins with the reverse transcription of RNA into complementary DNA (cDNA), followed by conventional PCR. RT-PCR is pivotal in gene expression studies and viral detection. (Gubler, et al., 1983) RT-PCR is a technique use in genetic studies that allows the detection and quantification of mRNA. Sensitive method that shows whether or not a specific gene is being expressed in a given sample. RT-PCR used to qualitatively detect gene expression through creation of complementary DNA transcript from RNA.
3. REAL TIME PCR:
Quantitative PCR, or real-time PCR, allows for the quantification of DNA during the PCR process. It provides insights into gene expression levels, making it crucial in genetic research and diagnostic applications. (Higuchi, et al., 1993) It monitors the amplification of a targeted DNA molecule during the PCR. Real-time PCR is carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of at least one specified wavelength and detect the fluorescence emitted by the excited fluorophore. The thermal cycler is also able to rapidly heat and chill samples, thereby taking advantage of the physicochemical properties of the nucleic acids and DNA polymerase.
4. NESTED PCR:
Nested PCR involves two sets of primers: an outer pair and an inner pair. The first PCR amplifies the target region, and the product is then used as a template for the second PCR with the inner primers. This technique enhances specificity and is valuable when working with complex samples. (Innis, et al., 1990)
5. MULTIPLEX PCR:
Multiplex PCR simultaneously amplifies multiple target sequences in a single reaction. It is time-efficient and cost-effective, making it advantageous in applications such as pathogen detection and genetic screening. (Gibson, et al., 1996) Amplifies multiple DNA template regions. Multiple pair of primers designed to target regions. Presence of multiple primers lead to cross hybridization.
6. HOTSTAR PCR:
Hot Start PCR utilizes a modified DNA polymerase that remains inactive until a specific temperature is reached. This prevents non-specific amplification and enhances the specificity of the reaction. (Sharkey, et al., 1994)
7. INVERSE PCR:
Inverse PCR is employed to amplify DNA fragments flanking a known sequence. It involves circularization of the target DNA, followed by PCR using primers facing outward. This technique is useful in genome walking and identifying unknown sequences. (Ochman, et al., 1988) Inverse PCR functions to clone sequences flanking a known sequence. Flanking DNA sequences are digested and then ligated to generate circular DNA. Amplification and identification of flanking sequence such as transposable elements and the identification of genomic inserts.
8. TOUCH DOWN PCR:
Touchdown PCR is a technique to limit non-specific amplification. Touchdown PCR offers a simple and rapid means to optimize PCR: Specificity, Sensitivity and Yield. To prevent lengthy optimization and primer redesigning.
CONCLUSION-TYPES OF POLYMERASE CHAIN REACTION:
In conclusion, the diverse types of PCR techniques have expanded the applications of this transformative technology. From conventional PCR for routine DNA amplification to specialized methods like qPCR for quantitative analysis, the evolution of PCR has revolutionized molecular biology, enabling breakthroughs in various scientific fields.
REFERENCES:
Mullis, K. B., et al. (1986). Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symposia on Quantitative Biology, 51(Pt 1), 263-273. https://pubmed.ncbi.nlm.nih.gov/3472723/
Gubler, U., et al. (1983). Reverse transcription of the RNA genome of influenza virus. Science, 222(4620), 551-553. https://pubmed.ncbi.nlm.nih.gov/9360371/
Higuchi, R., et al. (1993). Simultaneous amplification and detection of specific DNA sequences. Bio/Technology, 11(9), 1026-1030. https://pubmed.ncbi.nlm.nih.gov/1368485/
Gibson, U. E., et al. (1996). A novel method for real time quantitative RT-PCR. Genome Research, 6(10), 995-1001. https://pubmed.ncbi.nlm.nih.gov/8908519/
Sharkey, D. J., et al. (1994). Hot Start PCR: walking the line between specificity and simplicity. BioTechniques, 16(1), 113-117. https://pubmed.ncbi.nlm.nih.gov/8074881/
