PCR as a diagnostic tool
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What are primers?
They are ~20 nucleotide sequences designed to recognise and bind to specific target sequences.
What are the 5 components needed for PCR?
- Target - DNA in sample
- dNTP - nucleotides needed to make copies of DNA target
- Taq polymerase - enzyme which amplifies DNA at high temperature
- Buffer - to maintain appropriate reaction environment
Outline the steps involved in PCR
- Target DNA is denatured to separate the strands at 95°C
- Reaction is cooled to allow primers to anneal to the target at 55-65°C
- Primers then extended using Taq polymerase at 72°C
- This cycle is then repeated another 25-30 times
What extra component is added to reverse transcriptase PCR?
Reverse transcriptase - an enzyme that copies RNA to cDNA so it can be amplified in the PCR reaction
How is product normally detected using conventional or reverse transcriptase (RT) PCR?
Product is detected at the end of the reaction and is usually visualised on gel (load sample/dye into wells of gel and pass a current through the gel, fragments of different sizes travel different distances)
Describe some of the issues with conventional PCR
- PCR is very sensitive and so contamination of PCR reactions with PCR product must be avoided
- If the primers can bind elsewhere in the target nucleic acid sequence then the target sequence may not be amplified efficiently
- If there is sequence variation across the primer binding site mismatches can occur and the efficiency of the reaction may be lessened or no product may be amplified. This could lead to possible false negatives.
List some ways we can avoid cross contamination of PCR reactions with PCR products
- 'Clean' rooms for setting up of master mixes
- Addition of target in rooms where product is not amplified using equipment not used to handle the product
- Wearing of protective gear (clothes/gloves)
- Use of UV hoods which irradiate contaminating DNA/product
What is the difference between conventional and real time PCR (qPCR)?
Real time PCR allows product to be detected in real time rather than at the end of the PCR reaction. It also allows quantification of the target sample.
How is product detected in real time PCR?
Using a fluorescent dye
Real time PCR is a 'closed tube' reaction, what does this mean?
It means no product is released into the lab environment so there is decreased risk of contamination of later real time PCR reactions
The Ct (threshold cycle) is the cycle number at which sample fluorescence crosses threshold. In a real time PCR reaction, what is the significance of a low/high Ct?
- Low Ct = high amount of target in sample
- High Ct = low amount of target in sample
What are some of the veterinary uses of qPCR?
- Disease diagnosis - detection of pathogen genomes in the host
- Monitoring disease/infection process
- Screening to prevent introduction of an infection into a negative household/geographic area
- Research - gene expression and disease
What are the advantages and disadvantages of qPCR vs conventional PCR?
- Advantages - faster, more sensitive, more quantitative, safer (no ethidium bromide or radioactivity), more specific, possibly lower risk of contamination
- Disadvantages - More specialised equipment, more expensive, requires known sequence across primer and probe regions
Give examples of diseases that can be tested for using PCR
Canine parvovirus and feline herpesvirus (FHV)
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