What is the purpose of PCR?
Creating millions of copies of DNA for diagnostics, research, forensic medicine
What is the target?
DNA in the sample
What are primers?
Short sequences of DNA (oligonucleotides), designed to complement the sequence on the target DNA. 3’ and 5’.
What are dNTP?
Nucleotides used to create copies of DNA
What is taq polymerase?
Enyme which amplifies DNA at a high temperature
What is the purpose of a buffer?
To maintain an appropriate reaction environment
What are the 2 types of PCR?
Reverse transcriptase PCR (RT-PCR)
Real time PCR (qPCR)
What are the steps in RT-PCR?
- Mixture heated to 45-55 degrees for 15-30 mins to allow reverse transcriptase to act (unzip DNA helix)
- Heated to 95 degrees, for 15s, to denature the DNA
- Cooled to 60 degrees, for 30s, to allow primers to anneal to target DNA
- Heated to 72 degrees to extend the primers using taq polymerase
Cycle repeated 25-30 times
How are the strands of DNA seperated?
DNA is placed onto agarose gel. An electrical current is then passed through it causing DNA of different lengths to travel different distances through the gel.
How is the product of PCR verified?
Sequencing of product
Expected size of product
How can the product be used?
- Indicates presence of taret in sample - diagnosis in clinical case
- Analysis of product
- Cloning of product for use in research based/therapeutic based context
What are the precautions that need to be taken when carrying out RT-PCR?
- ‘Clean’ rooms for setting up of master mixes
- Addition of target rooms where product is not amplified
- Wearing of protective gear
- Use of UV hoods which irradiate contaminating DNA
What are issues with RT-PCR?
- Contamination of sample
- Specificity - primers can bind elsewhere in the sequence, target sequence may not be amplified efficiently
- Sequence variation across primer binding site - any mismatches across primer binding site means efficiency of reaction reduced (possible false negatives)
What is the difference between RT-PCR and qPCR?
qPCR - Amplified product is labelled with a fluorescent dye for detection. Product is detected in real time and is quantified.
What kinds of dye are used in qPCR?
- dsDNA intercalating dye - non-sequence specific, intercalate with DNA product
- Sequence specific probe - probes are attached to fluorochrome and bind to complementary DNA product
What are veterinary uses of PCR?
- Disease diagnosis
- Monitoring disease/infection progress
- Screening to prevent intrduction of infection into a negative area
- Research - gene expression and disease
What are the advantages to qPCR?
More sensitive More quantitative Faster Safer More specific Lowered risk of contamination
What are the disadvantages of qPCR?
Need more specialised equipment
More expensive
Requires known sequence across the primer and probe regions
What are the diagnostic advantages of PCR?
- Allows fast diagnosis of disease
2. Can identify the strain associated with the outbreak by using strain specific primers
What are the disadvantages to PCR?
- Requires a specialist lab
- Steps need to be taken to avoid contamination
- Existing primers may not recognise new strains of pathogen
What do vets need to be aware of?
- Make sure the test being used has been used in refereed publications
- Be clear about the purpose of your test
- Check to make sure the best sample is submitted
- Be aware of limitations of PCR
- The presence of an infectious organism doesn’t always mean that it is the cause of the disease
