Essential Biomedical Research Skills - Practicals (Strand A)

-Polymerase Chain Reaction -Amplification of DNA (exact copies) -Based on DNA polymerase being able to synthesise a new strand of DNA complementary to the original ‘template’ strand. -Starts with a 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.

• DNA helicase separates dsDNA strands, binds to structure and energetically breaks the H bonds between them. Phosphodiester bonds do not break. • Primases attach RNA primers to the open strands. • DNA polymerase binds to double stranded structure and slides along leading strand of DNA, producing complimentary strand, from 5’ to 3’. • Lagging strand, produce smaller fragments of DNA (Okazaki fragments) =pointless so ligase joins (ligates) these together.

Starts initial process on leading strand.

Produces lagging strand

Takes over to produce DNA product.

-Leading strand -Template DNA -Polymerases

• Sensitive – amplify as little as one molecule of DNA. • Specific – can amplify a unique target sequence, stringency depends on temperature and Mg2+. • Cheap • Rapid – results available in a few hours. • Robust – DNA is very stable and can be amplified from old and degraded samples.

-Template – double stranded DNA, the sequence to be amplified -2 primers – to prime synthesis in both directions (small ssDNA molecules, 6-30 bases, chemically synthesised) -Polymerase – copies the template, extending from 3’ end of primer. -dNTPS – deoxyribonucleotide triphosphates, dATP, dCTP, dGTP, dTTP -Magnesium – essential for polymerase. -Buffer – maintains optimal pH -Water - makes up final conc

-Building blocks -Phosphate (strength), sugar (orientation), base (specificity) -Thymine, Cytosine, Adenine, Guanine

-All living cells contain polymerase. -Copy DNA accurately -Enzyme has 3 regions: 1. Synthesis 2. Proof reading 3. Primer removal -Taq polymerase can withstand high temperatures.

-2 primers – one for each strand -Single stranded DNA (oligonucleotide) -Length of 18-24 bases: * Too long – hybridise too slowly. * Too short – won’t be specific, may bind elsewhere. -40-60% G/C content -Start and end with 1-2 G/C pairs. -Melting temp of 50-60C. -Primer pairs should have temp within 5C of each other. -3’ end must be complementary to the template DNA -Primer pairs should not have complementary regions.

-MgCl2 -A co-factor -A non-protein component of the reaction that’s needed to enable the activity of the catalysis. Maybe metallic or compound based. -Magnesium acts to enhance the enzymatic activity (specifically of DNA polymerase) thereby supporting DNA application.

-10x buffer -Optimal pH is 8-9.5 -Tris HCl -Potassium ions (KCl) – promotes annealing. *May be replaced by ammonium sulphate, which destabilises base pairing bonds.

-Denaturation – break bonds by heating to 90C -Annealing – primer binds to specific sites, temp reduced to 55C. -Elongation – heat to 72C, optimal temp for Taq polymerase to operate. 1st cycle – synthesis of a strand of DNA in a test tube 2nd cycle – synthesis of 2 strands in a test tube The rest – simultaneous synthesis of both strands -30 cycles = >1 billion copies

Holds the polymerase on the ssDNA.

• Clean gloves, lab coat, pipettes, and benches • Dedicated areas in lab • Care with tubes and templates • Don’t bring in old samples. • Use UV to ensure equipment isn’t contaminated. • Take your time, pipette accurately. • Pipette negative controls and samples first before positive controls.

• Able to amplify and manipulate DNA. • Good when DNA is scarce. • Diagnosis of genetic diseases. • Detect genetically modified material. • Ancient DNA • Forensic analysis of DNA samples • Manipulate DNA – create artificial fragments and then introduce into host cells and gets incorporated into genome (genetic modification) • Knock out genes – study gene function. • Fuse host proteins with GFP

End point: - Cheap - Semi quantitative at best – band intensity - Sequencing, genotyping, cloning - See results at end, plateau Real time: - More expensive - Quantity of PCR is proportional to amount of template - Quantification of gene expression, microarray verification, quality control and assay validation, SNP genotyping, copy number variation, viral quantification, siRNA /RNAi experiments - Measures at exponential phase – more precise

• ‘Housekeeping genes’ • Constant level of expression – not affected by experimental factors. • Essential to support validity of qPCR results. • Confirms RNA extraction was good and efficient. • Supports conclusions of expression levels (credibility)

• Gene expression (mRNA): diseased versus healthy/drug effects/environment changes • RNA virus infection levels

• Convert RNA (often mRNA) to cDNA. Use reverse transcriptase (PCR must start with DNA). • Amplify DNA by PCR (including qPCR)

In the reaction: - Thermos lightcycler - detects fluorescence - Fluorescent - SYBR green or TaqMan - Mastermix. • PCR product is measured as it is produced e.g., by incorporating fluorescent marker into the product. • The cycle number at which the fluorescence reaches a threshold value is measured. • The lower the Ct value, the greater the quantity of DNA/cDNA in the starting template

• Sensitive – can amplify as little as one molecule of DNA. • Specific – can amplify a unique target sequence stringency depend on temperature and Mg2+. • Cheap – relatively • Rapid – results available in a few hours. • Robust – DNA is very stable, can be amplified from old and degraded samples.

• Diagnosis of genetic traits • Detection of carriers of genetic traits • Tissue matching (HLA typing) • Predicting response to drugs (pharmacogenetics)