Principle of PCR
The PCR involves the primer mediated enzymatic amplification of DNA. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. Primer is needed because DNA polymerase can add a nucleotide only onto a preexisting 3′-OH group to add the first nucleotide. DNA polymerase then elongate its 3 end by adding more nucleotides to generate an extended region of double stranded DNA.
Components of PCR
The PCR reaction requires the following components:
- DNA Template : The double stranded DNA (dsDNA) of interest, separated from the sample.
- DNA Polymerase : Usually a thermostable Taq polymerase that does not rapidly denature at high temperatures (98°), and can function at a temperature optimum of about 70°C.
- Oligonucleotide primers : Short pieces of single stranded DNA (often 20-30 base pairs) which are complementary to the 3’ ends of the sense and anti-sense strands of the target sequence.
- Deoxynucleotide triphosphates : Single units of the bases A, T, G, and C (dATP, dTTP, dGTP, dCTP) provide the energy for polymerization and the building blocks for DNA synthesis.
- Buffer system : Includes magnesium and potassium to provide the optimal conditions for DNA denaturation and renaturation; also important for polymerase activity, stability and fidelity.
Procedure of PCR
All the PCR components are mixed together and are taken through series of 3 major cyclic reactions conducted in an automated, self-contained thermocycler machine.
- Denaturation :
This step involves heating the reaction mixture to 94°C for 15-30 seconds. During this, the double stranded DNA is denatured to single strands due to breakage in weak hydrogen bonds.
- Annealing :
The reaction temperature is rapidly lowered to 54-60°C for 20-40 seconds. This allows the primers to bind (anneal) to their complementary sequence in the template DNA.
- Elongation :
Also known at extension, this step usually occurs at 72-80°C (most commonly 72°C). In this step, the polymerase enzyme sequentially adds bases to the 3′ each primer, extending the DNA sequence in the 5′ to 3′ direction. Under optimal conditions, DNA polymerase will add about 1,000 bp/minute.
With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.
Types of PCR
In addition to the amplification of a target DNA sequence by the typical PCR procedures already described, several specialised types of PCR have been developed for specific applications.
- Real-time PCR
- Quantitative real time PCR (Q-RT PCR)
- Reverse Transcriptase PCR (RT-PCR)
- Multiplex PCR
- Nested PCR
- Long-range PCR
- Single-cell PCR
- Fast-cycling PCR
- Methylation-specific PCR (MSP)
- Hot start PCR
- High-fidelity PCR
- In situ PCR
- Variable Number of Tandem Repeats (VNTR) PCR
- Asymmetric PCR
- Repetitive sequence-based PCR
- Overlap extension PCR
- Assemble PCR
- Intersequence-specific PCR(ISSR)
- Ligation-mediated PCR
- Methylation –specifin PCR
- Miniprimer PCR
- Solid phase PCR
- Touch down PCR, etc
Applications of PCR
Some common applications of PCR in various fields can be explained in following categories.
1. Genetic testing for presence of genetic disease mutations. Eg: hemoglobinopathies, cystic fibrosis, other inborn errors of metabolism
2. Detection of disease causing genes in suspected parents who act as carriers.
3. Study of alteration to oncogenes may help in customization of therapy
4. Can also be used as part of a sensitive test for tissue typing, vital to organ transplantation
genotyping of embryo
5. Helps to monitor the gene in gene therapy
Infectious disease Applications:
1. Analyzing clinical specimens for the presence of infectious agents, including HIV, hepatitis, malaria, tuberulosis etc.
2. Detection of new virulent subtypes of organism that is responsible for epidemics.
1. Can be used as a tool in genetic fingerprinting. This technology can identify any one person from millions of others in case of : crime scence, rule out suspects during police investigation, paternity testing even in case of avaibility of very small amount of specimens ( stains of blood, semen, hair etc)
Research and Molecular Genetics:
1. In genomic studies: PCR helps to compare the genomes of two organisms and identify the difference between them.
2. In phylogenetic analysis. Minute quantities of DNA from any source such a fossilized material, hair, bones, mummified tissues.
3. In study of gene expression analysis, PCR based mutagenesis
4. In Human genome project for aim to complete mapping and understanding of all genes of human beings.