STRAND A Flashcards
no. H bonds between CG
3
no. H bonds between AT
2
primase function
synthesizes RNA primers
PCR benefits
sensitive
robust
cheap
rapid
specific
PCR tube contents
template (ds DNA)
2 primers
polymerase
dNTP’s
Magnesium
buffer (8-9.5)
3 regions of Taq polymerase
synthesis
proof-reading
primer removal
Taq polymerase characteristics
heat-stable
3 regions
accurate DNA copying
implications of too long of a primer in PCR
slow hybridization
implications of too short of a primer in PCR
not specific
primer size range in PCR
18-24 bp
primer characteristics in PCR
oligonucleotide/ ssDNA
start/ finish with G/C pairs
Tm = 50-60 degrees C (5 degrees between pairs)
3’ comp to template
Magnesium role in PCR
non-protein co-factor allowing catalysis for enzymatic activity of DNA polymerase
potassium ions role in PCR
promote annealing
PCR process 3 stages
denaturation
annealing
elongation
PCR 1st cycle
1 strand synthesis
(boil, anneal and extend w polymerase/ dNTPS)
PCR 2nd cycle
synthesis of 2 strands
(boiling and annealing different primer comp to new/original DNA strand, polymerase extends)
final PCR cycles
simultaneous synthesis of both strands
30 repeats
PCR product detection
molecular weight markers
PCR products
primers
template
(agarose gel w intercalating dye)
uses of PCR
DNA manipulation/ quantification/ amplification
genetic disease diagnosis
pathogen detection
ancient DNA
gene function study
knock-out genes
biotechnology
reverse transcriptase PCR
- RNA converted to cDNA via reverse transcriptase
- amplification via PCR
RNA sources in reverse transcriptase PCR
gene expression
RNA virus
reverse transcriptase PCR ingredients
reverse transcriptase
dNTPs
buffer
primer
RNA template
end-point vs qPCR
age?
price?
precision
qPCR newer (1996 vs 1983), more expensive, more precise
end-point vs qPCR quantification
end-point semi-quantitative, measuring densitometry
qPCR amount proportionate to template amount
end-point measurement at end (plateau) and qPCR continuous (exponential phase) measurement
end-point PCR uses
cloning
genotyping
sequencing
qPCR uses
gene expression
quantification
microarray verification
quality control assay validation
SNP genotyping
copy number variation
viral quantification
siRNA/ RNA experiments
THERMOS LIGHTCYCLER
thermal cycler incorporating fluorometer for detection and quantification of PCR products
master mix
pre-measured solution at optimal concentration for each reagent
2 fluorescence reagents
SYBR green
TAQman
SYBR green
binds to groove of dsDNA
TAQman
probes with fluorescence reporter and quencher
probe hybridizes as FUP/RUP anneal and extend
DNA pol cleaves probe and fluorescence increases
3 phases of PCR logarithmic standard curve
exponential
linear
plateau
Ct
cycle threshold
no. cycles required for the PCR to reach the threshold/ exceed background level.
Ct level indication
lower Ct value, higher amount of cDNA (starting material)
reference gene
control to normalize gene expression levels, constant gene expression, unaffected by experimental factors
house-keeping gene
normalizes mRNA levels between samples to for sensitive comparison
^reliability and reproducibility of experimental results
PCR standard curve
semi-log regression line plot of Ct value vs log of nucleic acid input
reference gene examples for PCR
Beta actin
GAPDH
Albumin
18S rRNA
TATA sequence binding protein
3 examples of clinical applications of PCR
Genotyping patient
genotyping pathogen
phenotyping disease
PCR patient genotyping components
genetic trait diagnosis
carrier detection
tissue matching (HLA typing)
predicting response to drugs
DNA sources for patient phenotyping
blood, hair, buccal smear, amniotic fluid cells
2 PCR based techniques for genotyping
PCR-RFLP (Restriction Fragment polymorphism)
ARMS-PCR (Amplification Refractory Mutation System)
PCR-RFLP process
- amplify substrate to 2 strands of dsDNA
- add the restriction enzyme
- analysis with electrophoresis
clinical example of PCR-RFLP
Diagnosis of Sorsby’s Fundus dystrophy
degenerative eye disease leading to blindness
autosomal dominant
TIMP3 mutation (tissue inhibitor of metalloproteinase 3) introduces premature stop codon
PCR RFLP positives
cheap
easy design
microindel/SNP application
simple resources
commonly used techniques
PCR RFLP negatives
only possible with known restriction site
some RE expensive
requires single nucleotide polymorphism
time-consuming
not suitable for high-throughput
clinical example of ARMS-PCR
diagnosis of cystic fibrosis
mutation in CFTR gene leading to Cl- transport imbalance across PM
F508 common mutation
ARMS-PCR
use of allele specific primers to detect alelic variants
RFLP vs ARMS
primers?
dependents?
alternatives?
RFLP has locus-specific primers vs ARMS allele specific primers
RFLP relies on presence/ absence of restriction site vs ARMS relies on PCR stringency
ARMS has tetra primer alternative w non-allele specific primers in addition
DNA sources for pathogen phenotyping
blood
sputum
urine
faeces
skin swab
tissue biopsy
pathogen phenotyping influence
patient management and infection control measures
microscopy disadvantages to PCR
less sensitive (high levels required)
difficult to distinguish strain/ species
culture disadvantages to PCR`
not all organisms can be cultured (PCR doesn’t require culture)
takes weeks (rather than hours)
less specific
patient antibody response disadvantages to PCR
May not illicit strong response whereas PCR not dependent on immune response
pathogen phenotyping clinical example
TB smear test w acid fast stain > dependent on bacterial load/ quality/ expertise
culture for mycobacteria then molecular testing
disease phenotyping technique
RT-PCR
clinical example of disease phenotyping
HIV viral load measurement with RT-PCR
