L5 - Clinical applications of PCR Flashcards
How many PCR cycles is optimal?
30-35
Any more & the enzyme becomes less efficient so gets broken down
What are the 3 steps of PCR?
DENATURE
95 degrees
ANNEAL
50-60 degrees
EXTENSION/ELONGATION
75 degrees
Why is PCR valuable?
Sensitive Specific Cheap Rapid Robust
Common uses of PCR in determining diagnosis & prognosis
Genotyping the patient
Genotyping the pathogen
Phenotyping the disease
Genotyping the patient
– Diagnosis of genetic traits
– Detection of carriers of genetic traits
– Tissue matching – HLA typing
– Predicting responses to drugs – pharmacogenetics
Genotyping the pathogen
– Diagnosis of species & strain of infecting pathogen
Phenotyping the disease
– Measuring disease progression
– Measuring disease severity
Sources of DNA when genotyping the patient?
- Blood
- Hair
- Buccal smear
- Cells from amniotic fluid
What are the 2 PCR-based techniques for genotyping an individual?
PCR-RFLP – Restriction Fragment Polymorphism
ARMS-PCR – Amplification Refractory Mutation System
What is an allele?
Any of the alternative forms of a gene that may occur at a given locus
What is a restriction enzyme?
An enzyme that digests (cuts) DNA at a highly specific site
PCR-RFLP – Restriction Fragment Polymorphism
The first step in a PCR-RFLP analysis is amplification of a fragment containing the variation
This is followed by treatment of the amplified fragment with an appropriate restriction enzyme
Since the presence or absence of the restriction enzyme recognition site results in the formation of restriction fragments of different sizes, allele identification can be done by electrophoretic resolvement of the fragments
Advantages of PCR-RFLP
- Cheap
- Easy design
- Applied to microindels & SNPs
- Simple resources
- Commonly used technique
Disadvantages of PCR-RFLP
- Only possible if the site contains a known RE site
- Some RE are expensive
- Only possible if a single nucleotide variation
- Hands on & time consuming
- Not suitable for high-throughput
ARMS-PCR - Amplification Refractory Mutation System
Detects allelic variants using allele-specific primers
Involves manipulation of the primer for the allele of interest
RFLP vs ARMS
Both relatively cheap
PCR-RFLP
• Uses locus specific primers
• Relies on the presence or absence of a restriction site to distinguish between variants
ARMS-PCR
• Uses allele specific primers
• Relies on the stringency of the PCR to distinguish between alleles
• Alternative is Tetra Primer ARMS-PCR which uses additional non-allele specific primers
Sources of DNA when genotyping the pathogen?
DNA has to of been exposed to pathogen
- Blood
- Sputum
- Urine
- Faeces
- Skin swab
- Tissue biopsy
What will the information from genotyping a pathogen influence?
Patient management – eg. choice of treatment
Infection control measures
Advantage of PCR over some other methods of microbial diagnosis
Sensitive – can detect single copy of genome
Specific – can identify species & strain
Sensitivity means no need for culture
PCR takes a few hours
Detects DNA/RNA therefore not dependent on the immune response
How does phenotyping the disease help us?
Can help us to tell:
• How severe is the disease?
• How is the disease likely to progress?
What is the key technique in disease phenotyping?
Quantitative PCR – measures the abundance of DNA or RNA in a clinical sample
To measure the level of infectious pathogen in a sample
To measure the level of expression of a gene (RT-PCR)
How do you measure the level of expression of a gene?
Using RT-PCR
To measure RNA by PCR, it must first be converted to cDNA by reverse transcriptase
The amount of DNA product after each cycle is proportional to the amount of RNA initially present
What are the 4 PCR techniques?
PCR-RFLP
ARMS-PCR
RT-PCR – PCR from RNA
Real-Time PCR – quantitative PCR
