CMB2000/L04 Types of PCR Flashcards
Name the reactants of PCR. (6)
DNA template
Primers
Nucleotides
Taq polymerase
Buffer
MgCl2
What is the role of MgCl2 in PCR?
Essential for Taq activity
[Mg2+] affects stringency of primer binding
Give the 3 steps of a PCR cycle.
Add excess primers & heat to separate strands
Cool to anneal primers
Synthesise new DNA
Give 3 common uses of PCR.
Genotyping the patient
Genotyping the pathogen
Phenotyping the disease
Describe genotyping the patient.
Detects which alleles an individual is carrying for a specific gene
Give 2 PCR-based techniques for genotyping an individual.
PCR-RFLP
ARMS-PCR
What is PCR-RFLP?
Restriction Fragment Polymorphism
Identifies allelic variants based on presence/absence of a restriction site
What are the 3 steps of PCR-RFLP?
Amplify
Cut PCR product with restriction enzyme R
Size-fractionate by gel electrophoresis
What would occur in PCR-RFLP if one allele has a restriction site for a specific restriction endonuclease and another allele did not?
RE site produces 3 products
a+b, a and b
What is the BamHI recognition site?
GGATCC
What is Sorsby’s Fundus Dystrophy?
Degenerative eye disease leading to blindness
Autosomal dominant
Mutation in TIMP3 (tissue inhibitor of metalloproteinase 3) gene introduces premature stop codon
Give 3 advantages of PCR-RFLP.
Cheap
Easy design
Applied to microindels and SNPs
Simple resources
Commonly used techniques
Give 3 disadvantages of PCR-RFLP.
Only possible if 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
Define genotyping the patient.
Determine which alleles an individual carries for a specific gene (or set of genes)
What 2 PCR techniques are used for genotyping an individual?
PCR-RFLP
ARMS-PCR
Describe ARMS-PCR (amplification refractory mutation system).
Simple method for detecting any mutation involving single base changes or small deletions present
OR
Absence of a PCR product diagnostic for the presence or absence of the target allele
Describe the process of ARMS-PCR. (2)
Design allele-specific primers
PCR with ASP1 or ASP2 + conserved primer
How are primers in ARMS-PCR different to normal PCR?
G-C at the end is not possible
Define cystic fibrosis.
Mutation in CFTR gene leads to imbalances in Cl- transport across plasma membrane
F508 mutation most common cause
Compare the primers used in PCR-RFLP and ARMS-PCR.
RFLP-PCR - uses locus specific primers
Relies on presence or absence of restriction site
ARMS-PCR - allele specific primers
Relies on stringency of PCR to distinguish between alleles
Give an alternative to ARMS-PCR.
Tetra Primer ARMS-PCR
Uses non-allele specific primers
Define genotyping the pathogen.
Identifies the species and strain of an infectious pathogen by isolating a specific gene/piece of DNA
What does the pathogen genotype influence in healthcare? (2)
Patient management e.g., choice of treatment
Infection control measures
Describe diagnosis of TB using pathogen genotyping.
Conclusive diagnosis depends on detection of TB in sputum
Previous depended on microscopy and culture
Can achieve same-day diagnosis using PCR
