Topic: Polymerase chain reaction, PCR
Question: Describe the steps in Polymerase Chain Reaction(PCR).Add notes on the clinical utility of PCR.
Click here for Reference Material-This material is informational alone and is not specifically prepared as an answer for any question. Readers may do their own research before finalising diagnoses according to the characteristics unique to each question. Readers should not proceed without cross-referencing with relevant textbooks as well as standard guidelines available.
Polymerase Chain Reaction or PCR is a technique used to amplify a specific DNA sequence in a sample. It involves the following steps:
1. DNA extraction: DNA is extracted from the sample containing the target DNA sequence. The DNA is purified away from other cellular components.
2. Primer selection: Short DNA oligonucleotide primers that are complementary to the target DNA sequence are selected. The primers flank the target region to be amplified.
3. DNA denaturation: The extracted DNA is heated to 95°C to denature the double stranded DNA into single strands.
4. Primer annealing: The temperature is lowered to 50-65°C to allow the primers to anneal to the single stranded DNA templates.
5. DNA extension: DNA polymerase catalyzes the addition of nucleotides to the 3′ end of the primers, synthesizing a new DNA strand.
6. Cycle repetition: Steps 3 to 5 are repeated for 30-50 cycles, doubling the DNA target region after each cycle. This results in rapid amplification of the target DNA region.
7. Final extension: A final extension step at 72°C is performed to ensure all strands are extended fully.
8. Holding: The amplified DNA samples are held at 4°C until they are analyzed or purified further.
Notes:
•PCR can produce over a million copies of target DNA from a single sequence.
•It has high sensitivity and specificity, but contamination risks must be minimized.
•PCR has widespread clinical utility for:
›Detecting infectious agents like bacteria, viruses in blood, tissues, etc.
›Detecting gene mutations and diagnosing genetic diseases›DNA fingerprinting and forensic analysis
›Measuring viral loads in chronic infections like HIV, HCV, etc.
›Detecting minimal residual disease and recurrence in cancers
›Rapid identification of pathogens during disease outbreaks
•Real-time PCR quantifies DNA amplification after each cycle, allowing initial template measurement.
Quantitative PCR (qPCR) is useful for viral load and gene expression analysis.
•Reverse transcriptase PCR (RT-PCR) first converts RNA into complementary DNA (cDNA) which is then amplified by PCR. Used to analyze gene expression.In summary, PCR is a versatile technique for amplifying specific DNA sequences. By understanding the steps involved and clinical applications of PCR, one can appreciate its utility as a diagnostic tool in modern medicine.
