PCR -L3 Flashcards
What are the uses of PCR in determining diagnosis and prognosis
Genotyping the patient, genotyping the pathogen, phenotyping the diseases
What does genotyping the patient mean
Detects which alleles an individual in carrying for a specific gene
What are the uses of genotyping the patient
Diagnosis of genetic traits, detection of carriers of genetic traits, tissue matching, predicting response to drugs
What are the sources of DNA for patient genotyping
blood, hair, buccal smear, cells from amniotic fluid
What are the 2 PCR based techniques for genotyping an individual?
PCR-RFLP and ARMS-PCR
What is PCR-RFLP?
Restriction fragment polymorphism.
What does PCR-RFLP do?
Identifies allelic variants based on presence/absence of restriction site
What are the steps in PCR-RFLP?
- amplify the substrate, 2. add the RE and then analyse via electrophoresis
What does it mean if RE is in both products?
Homozygous for the disease allele
What does it mean if RE is in neither products?
Homozygous for the healthy allele
What does it mean if RE is in one product?
heterozygous
Give a clinical example of a diagnosis via PCR-RFLP
Sorsby’s Fundus Dystrophy
What is the mutation in Sorsby’s Fundus Dystrophy
TIMP3 - premature stop codon.
Advantages of PCR-RFLP
Cheap
Easy design
Applied to microindels and SNPs
Simple resources
Commonly used techniques
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 and time consuming
Not suitable for high-throughput
What is ARMS-PCR
Amplification refractory mutation system
How does ARMS-PCR detect variation?
Detects allelic variants using allele-specific primers
Give a clinical example of a diagnosis via ARMS-PCR
Cystic Fibrosis
What is the mutation in CF?
CFTR gene = imbalance in Cl-
Most common CF mutation?
F508
What does primer A include in CF?
includes primer for mutant F508 deletion
What does primer B include in CF?
includes primer for wt at this position
Compare RFLP and ARMS
PCR-RFLP
* Uses locus specific primers (i.e. will amplify all variants of the chosen DNA
sequence)
* 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.
How is genotyping used in relation to pathogen?
Identifies the species and strain of an infectious pathogen (bacteria, virus or
parasite) by isolating a specific gene/piece of DNA
Where can DNA/RNA be obtained for pathogen genotyping
Blood, septum, urine, faeces, skin swab, tissue biopsy
What areas of treatment can be impacted by the information obtained from genotyping the pathogen
Patient management and infection control measures
What are the advantages of PCR > microscope
PCR:
Sensitive – can detect single
copy of genome
Specific – can identify species
and strain
Microscope:
Requires high levels of infecting
organism
Often difficult to distinguish different
species
Electron microscopy required to
visualise viruses
What are the advantages of PCR > culture
PCR:
Sensitivity means no need for culture. PCR shorter (few hours)
Culture:
Some org = cannot be cultured, can take weeks and hard to be strain specific
PCR vs Patient AB response
PCR:
Detects DNA/RNA therefore
not dependent on immune
response
Genotyping the pathogen
Patient Ab response:
Pathogen may not elicit a strong
antibody response
Give a clinical example of a diagnosis using pathogen genotyping
TB
Most common gene used in TB?
IS6110
What does diagnosis of TB depend on?
Conclusive diagnosis depends on detection of M. tuberculosis in sputum
Why do we phenotype the disease?
Shows us how severe it is and how likely disease is to proggress
What sort of PCR technique is used in phenotyping disease?
qPCR
Why do we use qPmCR?
measures the abundance of DNA or RNA in a clinical sample
What do we measure using qPCR?
to measure the level of infectious pathogen in a sample
to measure the level of expression of a gene
Phenotyping the disease – taking a
“snapshot” in time
What do we use to measure level of a gene exrpression?
RT-PCR
True or false: DNA and cDNA can be accurately quantified by real-Time qPCR
True
Give a clinical example of using RT-PCR
measurement of HIV viral load by quantitative RT-PCR
