Polymerase chain reaction PCR / Gene amplification
Unit 2 rDNA, PCR: Pharmaceutical Biotechnology Books, PDF Notes
- Gene amplification is obtaining multiple copies of a known DNA sequence/ a gene.
- Gene amplification can be done artificially, by using polymerase chain reaction (PCR) technique.
- Thus PCR is in vitro technique for gene amplification.
- It is a scientific technique in molecular biology used to generate billions of copies of a particular DNA sequence in short time.
- PCR was first developed by Kary Mullis in 1983.
Basic requirements for PCR technique
- A DNA segment (100-35,000 bp in length) to be amplified.
- Primers which are synthetic oligonucleotides of 17-30 nucleotides. They are complementary to the sequences present on the desired DNA segment.
- Four types of deoxyribonucleotides (dATP, dCTP, dGTP, dTTP). They are collectively called dNTPs.
- A thermo stable DNA polymerase that can withstand upto 94°C is used for polymerization of new DNA strand. Usually Taq polymerase isolated from bacterium Thermus aquaticus is used.
The vast majority of PCR method use thermal cycling i.e. alternately heating and cooling the PCR sample.
Essential steps of PCR technique
The step involves heating of DNA at about 91°C. The heating breaks the hydrogen bonds to make ssDNA i.e. single-stranded DNA. The DNA molecule with more G-C pairs needs a higher temperature.
It is pairing of primers to the ssDNA segment. The primers have to be designed as per the requirement. This step requires a temperature of about 55°C.
The temperature is raised to 72°C. The Taq polymerase adds dNTPs behind the primer on the ssDNA.
These three steps constitute one cycle of the reaction. The process is carried out for about 28 – 30 cycles beyond which its reliability decreases.
Each cycle of PCR takes about 3-5 minutes. As PCR progresses, the DNA generated is itself used as a template for replication, thus setting a chain reaction in which the DNA template is exponentially amplified. PCR is carried out on an automated machine.
Collecting DNA samples as evidence from small blood samples, semen, saliva, tissue, skin, etc.
Quick results, results from small amount of sample, more stable results.
- Medical and biological research for a variety of applications
- DNA cloning
- gene amplification
- functional analysis of genes
- diagnosis of hereditary disease
- DNA fingerprinting (used in forensic sciences and paternity testing)
- The detection and diagnosis of infectious diseases and diagnosis of cancer
- Study human evolution
- Clone DNA of Egyptian mummies
- In future, DNA can be used as unique identity card
SIMPLIFIED CONCEPTS OF BIOTECHNOLOGY By, Dr. Pramod Kadu & Ms. Suchita Vishwakarma
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