PCR Test (Polymerase Chain Reaction)
PCR Test, The polymerase chain reaction (PCR) is a powerful method for the rapid amplification of deoxyribonucleic and (BNA) techniques was developed by Dr. K.B. Malik for which he received the Nobel prize in 1993. The PLR technique employs the enzyme DNA polymerase to multiply selectively a single molecule of DNA several million folds in few a hours. It is frequently used to detect small quantities of DNA specific to a pathogenic agent in blood or other body fluids. The polymerase chain reaction is a method widely used to rapidly make millions to billions of copies of a specific DNA sample. The tests work by finding the DNA or RNA of a pathogen (disease-causing organism) or abnormal cells in a sample.
DNA is the genetic material that contains instructions and information for all living things.
RNA is another type of genetic material. It contains information that has been copied from DNA and is involved in making proteins.
Principle of PCR Test:
Polymerase chain reaction (PCR) is a technology used for quick and easy amplifying DNA sequences, which is based on the principle of enzymatic replication of nucleic acids. This method has in the field of molecular biology irreplaceable role and constitutes one of the basic methods for DNA analysis.
Types OF PCR:
- Real-time PCR
- Quantitative real-time (Q-RT PCR)
- Reverse Transcriptase PCR
- Multiplex PCR
- Nested PCR
- Arbitrary primed PCR
- Methylation-specific RCR (MSP)
- Asymmetric PCR
- Assemble PCR
- ligation-mediated PCR
- Fast-Cycling PCR
- Long-range PCR
Components Of PCR:
The components of PCR constitute the following.
The DNA interest from the sample.
Taq polymerase is used.
Single-stranded DNA is complementary to the 3 ends of sense and anti-sense strands.
Deoxyribonucleotide triphosphate: (ddNTPs)
These provide energy for polymerization and are building blocks for the synthesis of DNA. These are single units of bases.
Magnesium and potassium provide optimum conditions for DNA denaturation and renaturation. It is also important for fidelity, polymerase activity, and stability.
Procedure / Steps of PCR:
Collect the 3 to 5ml blood samples in EDTA or red top Tube. Centrifuge the blood sample and separate the plasma or serum or plasma and serum or plasma used for the PCR test. The first step is Denaturation. The DNA template is heated to 94°C. This breaks the weak hydrogen bonds that hold DNA strands together in a helix, allowing the strands to separate creating single-stranded DNA.
The mixture is cooled anywhere from 50-70°C This allows the primers to bind (anneal) to their complementary Sequence in the template DNA- AT 45sec. It is a repeated process.
The reaction is then heated to 72°c the temperature for DNA polymerase to act DNA polymerase. optimal extends the primer, adding nucleotides onto the primer in a sequential manner, using the target DNA as a template. With one cycle, a single segment of double-stranded DNA template is amplified into separate pieces of double-stranded DNA. These two places are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the member of copies of the template is increased exponentially.
Application of PCR:
The following are the applications of PCR:
- Forensic Science
- Research and Genetics (Gene mapping, gene expression)
Advantages of PCR:
PCR is so sensitive that the DNA present in an individual cell can be isolated and amplified. This process is faster and less tedious than the traditional methods of gene cloning.