types of PCR Flashcards
lecture 4
What are the key components needed in a PCR reaction tube?
DNA Template – Sequence to be amplified.
Primers – Forward and reverse to start new DNA synthesis.
Nucleotides – Building blocks (dATP, dCTP, dGTP, dTTP).
Taq Polymerase – Catalyzes DNA synthesis.
Buffer (Tris-HCl, pH 8.0) – Maintains optimal pH.
MgCl₂ – Essential for Taq activity and primer binding stringency.
Why is PCR valuable in clinical settings?
Sensitivity – Can amplify as little as one DNA molecule.
Specificity – Amplifies unique target sequences.
Cost-Effective – Relatively inexpensive.
Rapid – Results in a few hours.
Robust – Works with degraded or old DNA samples.
What are common clinical uses of PCR?
Diagnosing genetic traits.
Detecting carriers of genetic disorders.
Tissue matching (HLA typing).
Pharmacogenetics (predicting drug responses).
Measuring disease progression/severity.
Identifying species/strains of pathogens.
What are two PCR-based genotyping techniques?
PCR-RFLP – Detects allelic variants by restriction site presence/absence.
**ARMS-PCR **– Uses allele-specific primers to detect specific alleles.
How does PCR-RFLP work?
Amplify DNA sequence with primers.
Use restriction enzymes to cut DNA at specific sites.
Analyze fragments using gel electrophoresis.
Identify alleles based on fragment pattern:
- Homozygous Healthy: No cut site.
- Homozygous Diseased: Two fragments.
- Heterozygous: Mixed fragments.
Clinical example of PCR-RFLP: Diagnosis of Sorsby’s Fundus Dystrophy.
Disease: Degenerative eye disease caused by a TIMP3 mutation.
Mutation introduces premature stop codon.
PCR amplifies region, SpeI restriction enzyme identifies mutation.
Gel shows distinct fragment patterns for wild-type and mutated alleles.
What is ARMS-PCR, and how does it differ from PCR-RFLP?
ARMS-PCR: Uses allele-specific primers to detect point mutations.
Differences:
Does not rely on restriction sites.
Relies on primer specificity for alleles.
Clinical example of ARMS-PCR: Diagnosis of Cystic Fibrosis.
Mutation: F508 deletion in the CFTR gene.
Primers are designed for:
Wild-type allele.
Mutant allele.
Gel electrophoresis distinguishes healthy, carrier, or diseased states.
What are the advantages of PCR for pathogen genotyping?
Sensitive – Detects single DNA/RNA copies.
Specific – Identifies species and strains.
Rapid – Results in hours, no culture needed.
Clinical example: PCR for Tuberculosis diagnosis.
Detects Mycobacterium tuberculosis DNA in sputum samples.
PCR produces a 439 bp product specific to the pathogen.
Same-day diagnosis possible, unlike slower traditional methods.
What is phenotyping in PCR, and how is it performed?
Measures disease status at a specific time.
Quantitative PCR (qPCR) is used to:
Measure pathogen levels (viral load).
Assess gene expression (mRNA to cDNA with RT-PCR).
How does quantitative PCR (qPCR) measure DNA?
Uses fluorescent dyes like SYBR Green.
Tracks DNA amplification cycle-by-cycle.
Fluorescence indicates DNA product accumulation.
Cycle Threshold (Ct):
The cycle when fluorescence surpasses a detectable threshold.
Lower Ct = Higher starting DNA concentration.
Clinical example: Measuring HIV viral load using qPCR.
RNA is converted to cDNA using RT-PCR.
qPCR quantifies HIV RNA levels.
Useful for monitoring disease progression and drug therapy response.
Summary of PCR applications in clinical labs.
Diagnosis: Genetic disorders, infectious diseases.
Prognosis: Disease monitoring, progression, and severity.
Techniques:
PCR-RFLP.
ARMS-PCR.
RT-PCR (from RNA).
qPCR (quantitative).
PCR-RFLP pros and cons
Advantages: Simple, cost-effective, reliable for small-scale analyses.
Disadvantages: Limited to sites with restriction enzyme recognition; labor-intensive.
