PCR

-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

• 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

• 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

Makes RNA primers that attach to the template

Holds the polymerase on the ssDNA

• 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

• 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

• Optimal pH = 8-9.5 • Tris HCl • Potassium ions (KCl), promotes annealing • ^ can be replaced by ammonium sulphate, destabilises base pairing bonds

• Clean gloves, lab coat, pipettes, benches • Care with tubes, templates • Don’t bring in old PCR samples • Use UV to avoid contamination • Pipette accurately

• Amplifies DNA, good when DNA scarce • Manipulate DNA, Forensic analysis, ancient DNA • Detect pathogens, diagnose genetic diseases, detect genetically modified material

• 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)

Detects which alleles an individual carries for a specific gene(s)

Blood, hair, buccal smear, cells from amniotic fluid

• PCR-RFLP (Restriction Fragment Polymorphism) • ARMS-PCR (Amplification Refractory Mutation System)

Alternative forms of a gene that may occur at a given locus

Enzyme that digests/cuts DNA at a highly specific site

+ 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

Identiifes the species and strain of an infectious pathogen by isolating a specific gene/piece of DNA

Blood, sputum, urine, faeces, skin swab, tissue biopsy