Describe PCR in basic terms
-Amplification of DNA
-DNA polymerase synthesis new strand of DNA complementary to original ‘template’ strand
-Starts with single stranded piece of DNA
-Uses Taq polymerase for repeated ‘cycles’
-With each cycle there is an exponential increase in strands
-Essentially copies aspect of replication
Why use PCR
- Sensitive - as little as one molecules of DNA
- Specific - a unique target sequence stringency depends on temperature and magnesium
- Cheap(ish)
- Rapid - few hours
- Robust - DNA is very stable, can be amplified from old and degraded samples
What goes into PCR tube?
- Template - double stranded DNA
- 2 primers - to prime synthesis
- Polymerase - copies template from 3’ end of primer
- dNTPs - dATP, dCTP, dGTP, dTTP
- Magnesium - co-factor for DNA polymerase
- Buffer - maintains pH, provides necessary salt
- Water - makes up final conc
What does primase do?
Makes RNA primers that attach to the template
What does the sliding clamp do?
Holds the polymerase on the ssDNA
Properties of primers
- 2, one for each strand
- 18-24 bases: too long = hybridise too slowly, too short = wont be specific, likely to bind elsewhere
- 40/60% G/C content
- Start and end with 1-2 G/C pairs
- Melting temp = 50-60C
- Primer pairs should have a temp within 5C of each other
- 3’ end must be complementary to template DNA
- Primer pairs should not have complementary regions
Features on MgCl2 in PCR
- A co-factor
- Non-protein component of the reaction that’s needed to enable the activity of the catalysis
- Acts to enhance the enzymatic activity, supporting DNA application
Properties of buffer (10x)
- Optimal pH = 8-9.5
- Tris HCl
- Potassium ions (KCl), promotes annealing
- ^ can be replaced by ammonium sulphate, destabilises base pairing bonds
What makes a good PCR?
- Clean gloves, lab coat, pipettes, benches
- Care with tubes, templates
- Don’t bring in old PCR samples
- Use UV to avoid contamination
- Pipette accurately
Why is PCR important/useful?
- Amplifies DNA, good when DNA scarce
- Manipulate DNA, Forensic analysis, ancient DNA
- Detect pathogens, diagnose genetic diseases, detect genetically modified material
What is reverse transcriptase PCR?
- Convert RNA to cDNA. Use reverse transcriptase, retroviral enzyme, that converts RNA to DNA (PCR must start with DNA)
- Amplify DNA by PCR (including qPCR)
Purpose of genotyping the patient?
Detects which alleles an individual carries for a specific gene(s)
Sources of DNA when genotyping the patient
Blood, hair, buccal smear, cells from amniotic fluid
2 PCR techniques for genotyping an individual…?
- PCR-RFLP (Restriction Fragment Polymorphism)
- ARMS-PCR (Amplification Refractory Mutation System)
What is an allele?
Alternative forms of a gene that may occur at a given locus
What is a restriction enzyme?
Enzyme that digests/cuts DNA at a highly specific site
Advantages and disadvantages of PCR-RFLP
+ cheap, easy, simple resources, commonly used techniques, applied to microindels and SNPs
- site needs RE site, RE can be expensive, time consuming, only possible if single nucleotide variation
Purpose of genotyping the pathogen
Identiifes the species and strain of an infectious pathogen by isolating a specific gene/piece of DNA
Where is DNA obtained from when genotyping the pathogen?
Blood, sputum, urine, faeces, skin swab, tissue biopsy
