FISH 543 / OCEAN 575 Molecular Techniques PCR FISH 543 / OCEAN 575 Molecular Techniques

DNA Replication in the Tube PCR Polymerase Chain Reaction Most important recent discovery (1985) Patented – all PCR reactions pay royalty Repeated replication of specific DNA sections Small quantities Feathers, hair etc. Specific regions of DNA Target specific sequences Logarithmic replication 2  4  8  16  32  64 128  256  512  1028

PCR How does it work: Separate the two strands (94oC) Anneal primers (55oC) Replication start Extension (72oC) = replication Repeat 20 – 30 times 94° 94° 72° 55°

PCR

PCR in practice Reaction ingredients Buffer Keep pH constant Template DNA Primers As a starting point Forward and reverse Nucleotides To synthesize DNA Polymerase Taq polymerase MgCl2 Aids enzyme activity Needs accurate temperature control PCR machines Automatic cycling of temperature

DNA Replication in the Tube PCR Need PCR primers Polymerase can only start synthesizing from double stranded DNA Start where primer anneal What are primers? Short artificial DNA sequences 15-20 bp Match template DNA Can pick where we want to start PCR Which direction?

The structure of DNA Sugar-phosphate backbone Nitrogenous base 5 C-atoms in the sugar Chain is directional #3 on one side #5 on the other Nitrogenous base Purines: A, G Pyrimidines: C, T Purines Pyrimidines

The structure of DNA Complimentary binding Hydrogen bonds Purine with Pyrimidine A – T G – C Chain is antiparallel

Action of DNA polymerase is always 5’ 3’

DNA sequences are always written 5’ 3’ 5’-GCCATAGATGCAGCCTGAGATCAGCATGCA-3’ 3’-CGGTATCTACGTCGGACTCTAGTCGTACGT-5’ 5’-GCCATAGATGCAGCCTGAGATCAGCATGCA-3’ 3’-ACGT-5’ 5’-GCCA-3’ 3’-CGGTATCTACGTCGGACTCTAGTCGTACGT-5’ So the Primers are 5’-GCCA-3’ and 5’-TGCA-3’

PCR primers Annealing temperature Remember: Optimal temperature for primers to attach to the template DNA Too high Bonds don’t work Primer doesn’t anneal Too low Primer may attach anywhere ‘Non-specific amplification’ Depends on strength of bonds Remember: G-C – three hydrogen bonds A-T – two hydrogen bonds Annealing temperature depends on GC content

Primers Where do we get primer sequences from? Somebody may have isolated them Check databases Freely available on internet (GenBank) Results not publishable without primer information Heterologous primers Isolated from related species Very useful for many applications Problem may not exactly match PCR does not always work Primer design from published sequences Align related species Design primers in conserved regions Amplify variable regions Primer isolation Very lengthy and expensive procedure several months work

Primer design Primer pairs should have similar annealing temp length, %GC content Tm = 4(G + C) + 2(A + T) oC. Primers should have no self complementarity 5’-ACTGT AGAT-3 GCC ATA GGC Minimal (<3bp) between-primer-complementarity 5’-ACTGTGCCATAGATGCAG-3’ |||| 3’-CAACTGCACCGTATGCAT-5’ Programs on the web to design primers Links on webpage

PCR - in practice Sample Single Reaction Template DNA 1-2 µg genomic 1-2 µg mtDNA 1µl Forward Primer 10 mM 2.5 µl Reverse Primer 10 mM 2.5 µl dNTPS 8mM 2.5 µl Mg++ 20mM 2.5 µl 10X buffer 2.5 µl H2O 11.5 µl Taq 0.5 U >1 µl Total 25 µl Primers, dNTPS and Mg are often made up as 10X stocks for ease of setting up reactions Buffer is polymerase-specific, purchased with the enzyme, Caution: some buffers are Mg++ free, others are not Use high quality nuclease free water

PCR - in practice You are never setting up only a single PCR reaction Make up master mix Buffer, primers, MgCl2, water, dNTPs, Taq When calculating master mix volume, add a bit (~1 sample’s worth) extra to allow for pipetting errors Negative control No template DNA Check for contamination Positive control Something you know works

Common PCR Problems Contamination No or weak product Primer dimers Non-specific products

The worst problem – Contamination Exponential copying of template Very sensitive Tiny amounts of contaminant can cause problems Main culprit PCR products Perfectly matching short sequences Massive amounts Can swamp new template DNA You are your own worst enemy! Solutions Use ultra-clean chemicals Separate pre- and post PCR Always use negative control Aliquot reagents in small batches Can be discarded if problem Use filtertips Pipet carefully

If it happens… Try somebody else’s ingredients Change ingredients chemicals water Clean gear pipettes bench (bleach) Be more careful Pipetting Use of contaminated tips Causes chemical contamination

No or weak product Missing ingredient Wrong concentrations Check your lab book Do it again Wrong concentrations Template Primer Taq MgCl2 Wrong primers Check sequence Try alternatives Use positive control Bad template Check template on agarose gel Fragmentation PCR inhibitors Add to working PCR Too much Wrong conditions Reduce stringency Reduce annealing temp Increase MgCl2 Failed staining Check visualization Use standard

Primer dimers Primers annealing to each other Small products 50-100 bp Usually because of template problems Primers try to anneal to something Solution Positive control Redesign primers Hot Start

Non-specific products Detection Electrophoresis on a gel Wrong product size Always use a standard Know your size Solution Increase stringency Increase annealing temperature Reduce MgCl2 Change program Extension times Different primers Reduce number of cycles

Amount of PCR product Number of PCR cycles Desired product Non-specific product Amount of PCR product Non-specific product with higher amplification efficiency than desired product Number of PCR cycles

PCR optimization Very sensitive procedure Each primer pair needs to be optimized Can vary between PCR machines Usually need to be optimized Concentrations MgCl2 conc Primer & template concentration Template can inhibit PCR - dilute Ratio often important dNTP conc Cycling parameters Annealing temp Based on primer Tm Extension times Potentially lots of variables Ways to make it easier Gradient cycles Allow annealing temp gradient across the block Can vary MgCl2 at same time Touch-down PCR Start with high annealing temp Produce few very specific copies Lower annealing temp More efficient replication Touch-up PCR Start with low annealing temp Make sure there are some copies Increase annealing temp Primers prefer PCR products Prevents non-specific amplification after many cycles

PCR optimization - rules Maximize stringency Highest annealing temp Lowest MgCl2 Minimize number of cycles Taq degradation Production of non-specifics Taq errors Most significant parameters Annealing temperature MgCl2