Month: December 2020
A Brief Intro To The Universal – PCR Technique
PCR (Polymerase chain reaction ) technology has revolutionised the practice of science and is the epic of the current explosion of technology in molecular biology. Since its invention in 1985 by K. Mullis and co-workers, PCR technique has not only found its application in research labs but has been widely used in clinical diagnosis, e.g., diagnosing disease, predicting disease course, and identifying infectious agents.
PCR has made it possible to obtain millions of copies of a DNA fragment from an extract, by in vitro replication. Matrix DNA can not only be a genomic DNA but it can also be a complementary DNA obtained by RT-PCR from a messenger RNA extract (poly-A RNA), as well as mitochondrial DNA. It is a technique for obtaining large amounts of a specific DNA sequence from a small DNA sample. This amplification is based on the principle of replication of a double-stranded DNA template. The process can be divided into three phases: a denaturation phase, a hybridization phase with primers, and an elongation phase (Fig.). The products of each synthesis step serve as a template for the following steps, thus exponential amplification is achieved.
PCR has revolutionized molecular medicine and molecular biology. Major research areas, such as gene regulation, biomarker discovery and cancer research, are challenging today’s PCR technologies with more demanding requirements. These include the need for higher assay sensitivity, increased throughput, and reliable data analysis. Assay development and evaluation, time to result and reproducibility of data, are still major problems encountered by researchers.
There are different types of PCR based on the markers used, conditions set, and the region of expansion.
- Amplified fragment length polymorphism (AFLP) PCR
- Allele-specific PCR
- Alu PCR
- Assembly PCR
- Asymmetric PCR
- COLD PCR
- Colony PCR
- Conventional PCR
- Digital PCR (dPCR)
- Fast-cycling PCR
- High-fidelity PCR
- High-Resolution Melt (HRM) PCR
- Hot-start PCR
- In situ PCR
- Intersequence-specific (ISSR) PCR
- Inverse PCR
- LATE (linear after the exponential) PCR
- Ligation-mediated PCR
- Long-range PCR
- Methylation-specific PCR (MSP)
- Miniprimer PCR
- Multiplex-PCR
- Nanoparticle-Assisted PCR (nanoPCR)
- Nested PCR
- Overlap extension PCR
- Real-Time PCR (quantitative PCR or qPCR)
- Repetitive sequence-based PCR
- Reverse-Transcriptase (RT-PCR)
- Reverse-Transcriptase Real-Time PCR (RT-qPCR)
- RNase H-dependent PCR (rhPCR)
- Single cell PCR
- Single Specific Primer-PCR (SSP-PCR)
- Solid phase PCR
- Suicide PCR
- Thermal asymmetric interlaced PCR (TAIL-PCR)
- Touch down (TD) PCR
- Variable Number of Tandem Repeats (VNTR) PCR
PCR is both a thermodynamic and an enzymatic process, so proper knowledge of it is required to deal with the trouble shooting areas and get good results yet few individuals who are currently using this technology, received detailed formal training in this field. This course is specifically designed keeping all these in mind, to educate and train individuals in the use of PCR technology and introducing one to the different types of PCR as well.
Importance Of This Course:
At the end of this course, the participant will be able to:
- Explain the basic steps involved in PCR
- Identify the components of PCR and PCR product analysis
- Recognize the characteristics of different PCR techniques
- Identify the techniques used to detect products in real-time PCR
- Differentiate the nucleic acid quantification processes used in real-time PCR
- Explain the roles of PCR controls
- Learn about multiplex PCR
- Tell the pitfalls of PCR as a research tool
- How to overcome the problems faced while doing PCR
Benefits in career:
The polymerase chain reaction has been elaborated in many ways since its introduction and is now commonly used for a wide variety of applications including genotyping, cloning, mutation detection, sequencing, microarrays, forensics, and paternity testing.
So, those who are taking this course will find wide application in research work.