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.
Buffers.

The vast majority of PCR method use thermal cycling i.e. alternately heating and cooling the PCR sample.

Essential steps of PCR technique

Heat denaturation

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.

Annealing

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.

Polymerization

The temperature is raised to 72°C. The Taq polymerase adds dNTPs behind the primer on the ssDNA.

Diagrammatic representation of steps involved in PCR technique

Key points

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.

Advantages

Quick results, results from small amount of sample, more stable results.

Uses

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

Reference:

SIMPLIFIED CONCEPTS OF BIOTECHNOLOGY By, Dr. Pramod Kadu & Ms. Suchita Vishwakarma

Third Year B Pharm Notes, Syllabus, Books, PDF Subjectwise/Topicwise

T Y B Pharm Sem V	T Y B Pharm Sem VI
BP501T Medicinal Chemistry II Theory	BP601T Medicinal Chemistry III Theory
BP502T Industrial Pharmacy Theory	BP602T Pharmacology III Theory
BP503T Pharmacology II Theory	BP603T Herbal Drug Technology Theory
BP504T Pharmacognosy II Theory	BP604T Biopharmaceutics and Pharmacokinetics Theory
BP505T Pharmaceutical Jurisprudence Theory	BP605T Pharmaceutical Biotechnology – Theory
BP506P Industrial Pharmacy I Practical	BP606T Quality Assurance Theory
BP507P Pharmacology II Practical	BP607P Medicinal chemistry III Practical
BP508P Pharmacognosy II Practical	BP608P Pharmacology III Practical
	BP609P Herbal Drug Technology Practical

Polymerase chain reaction PCR / Gene amplification