🐭 RAT series
Points to Ponder for NEET-PG, FMGE & NEXT
- PCR is a powerful technique for amplifying specific DNA segments.
- Specificity relies on complementary primers flanking the target sequence.
- Taq polymerase and thermal cycling are essential for exponential amplification.
- PCR has numerous applications in medicine, research, and forensics.
PCR is a fundamental laboratory technique used to amplify (make millions of copies) of a specific DNA segment. It is a powerful tool in various fields, including medicine, forensics, and research.
Principles:
- Specificity: Achieved by using two short, single-stranded DNA molecules called primers that are complementary to opposite ends of the target DNA sequence.
- Enzymatic amplification: Utilizes a heat-stable DNA polymerase (Taq polymerase) to synthesize new DNA strands complementary to the target sequence and primers.
- Thermal cycling: Repeated cycles of heating, annealing, and extension create an exponential increase in the target DNA copies.
Steps:
- Denaturation: High temperature (>90°C) separates the double-stranded DNA, creating single strands.
- Annealing: Primers complementary to the target sequence hybridize (bind) to each single strand at a lower temperature (50-75°C).
- Extension: Taq polymerase extends the primers using free nucleotides (dNTPs) to generate new DNA strands complementary to the target sequence.
- Cycle repetition: Steps 1-3 are repeated for 20-40 cycles, resulting in exponential amplification of the target DNA.
Key components:
- Taq polymerase: A heat-stable DNA polymerase isolated from Thermus aquaticus bacteria, allowing high-temperature denaturation steps.
- Deoxyribonucleotides (dNTPs): The building blocks (A, T, C, G) for synthesizing new DNA strands.
- Magnesium (Mn2+): Essential cofactor for Taq polymerase activity.
Applications:
- Infectious disease diagnosis: Detecting and quantifying pathogens like viruses and bacteria.
- Prenatal genetic diagnosis: Identifying genetic disorders in a fetus before birth.
- Tissue typing: Matching donor and recipient tissues for transplantation.
- Quantitative gene expression analysis (RT-PCR): Measuring the amount of specific RNA molecules in a sample (requires reverse transcription to convert RNA to complementary DNA, cDNA).
