PCR

1. PCR FISH 543 / OCEAN 575 Molecular Techniques

2. DNA Replication in the TubePCR • 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

3. 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°

4. PCR

5. PCR in practice • Needs accurate temperature control • PCR machines • Automatic cycling of temperature • 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

6. DNA Replication in the TubePCR • 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?

7. The structure of DNA • Sugar-phosphate backbone • 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

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

9. Action of DNA polymerase is always 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’

10. 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’ 5’-TGCA-3’ and

11. PCR primers • Annealing temperature • 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 dependson GC content

12. 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

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

14. 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++ 20mM2.5 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

15. 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

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

17. 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

18. 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

19. Missing ingredient 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 No or weak product

20. 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

21. 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

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

23. 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

24. 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 PCR optimization - rules