Contents Hospital PCR gel Results Eppendorf EB gel K Mullis Big issue PCR Summary How much DNA? How many cycles? Apparatus Taq, PFU Glossary Cycling parameters 3’ end for specificity Bad primers Hot start Nested primers Alder

Interpretation of DNA tests There was a fire in the maternity Hospital and the mothers and new babies had to be evacuated. After the fire was out and all the mums and babies were safely back in the wards, one mother insisted that the baby boy she had been given was NOT hers. DNA was isolated from samples taken from this mother and the father of her baby, and also from the five boys in the ward. A very small fragment of the DNA was then amplified (copied) using PCR (Polymerase Chain Reaction). The fragment is used for forensic tests because its size is variable and because it is present in two copies, one from each parent, in all humans. The variability is caused by internal duplication of a 16 bp (base pair) sequence of DNA. The 16 bp sequence is repeated between 14 and 41 times in different peoples genomes. (All but the 15-mer repeats occur frequently.) Can you discover which of the boys belongs to the parents using the test results shown in the figure blow ?

Agarose gel for separating DNA fragments according to size Allelic ladder Mother Father Boys 1 2 3 4 5 large DNA fragments small DNA - Migration of DNA + Which boy belongs to the mother and father?

Allelic ladder Mother Father Boys 1 2 3 4 5

Polymerase Chain Reaction PCR Polymerase Chain Reaction Kary B Mullis Method for amplifying DNA fragments In vitro alternative to DNA cloning

240 = 1 trillion 1012 Day 0 Investment Day 1 Profit Double investment every day 10 days 20 days 30 days 40 days 210 = £ 1024 (103) 220 = £ 1 million (106) 230 = £ 1 billion (109) 240 = 1 trillion 1012

95 C DNA polymerase dNTPs Primer 1 5’ dNTPs ss DNA, “Template” 3’ PCR reactions 1 and 2 DNA polymerase dNTPs Primer 1 5’ 95 C Primers 1+2 DNA polymerase dNTPs ss DNA, “Template” 3’ known sequence (to denature DNA) known sequence 2 partially double-stranded DNA molecules Primer 2

95 C dNTPs PCR reactions 3 Primers 1+2 DNA polymerase PCR fragment dsDNA, specific lenght partially ds DNA

dsDNA Summary PCR reactions 1-3 n = 2 i-2 (+2 i-3) Template Primers 1+2 dNTPs 3x 95 C denaturation 3x DNA polymerase 2 dsDNA PCR fragments of specific length From now on... Every denaturation-polymerisation cycle (i) doubles the number (n) of PCR fragments n = 2 i-2 (+2 i-3) …………. until dNTPs or primers run out, or the enzyme activity becomes limiting.

How much DNA can be synthesised ? 100 µl PCR reaction Ingredient pmoles Molecules Capacity dNTPs 800 5•1014 0.4 µg dsDNA Primers 2 x 50 2 x 3•1013 33 µg 1 kb DNA dNTPs are limiting 0.4 µg dsDNA 1 kb fragment = 0.7 pmoles = 4x1011 molecules - sufficient for 10 strong bands on agarose gel - sufficient for several cloning experiments How many PCR cycles starting from a single template molecule ? 40+2 cycles (c. 4 h in PCR machine)

Running too many PCR cycles creates artefacts! How many PCR cycles are required to synthesise 1 g DNA from 1 ng of template DNA? Running too many PCR cycles creates artefacts! 1 pmole = 6•1011 molecules

Detection and identification of pathogenic bacteria PCR Applications of PCR DNA fingerprinting RFLP mapping Detection and identification of pathogenic bacteria DNA for sequencing and cloning Joining sequence contigs “gap filling” Contig 1 Gap <10 kb Contig 2

Heat-stable DNA polymerases for PCR Elongation at 72 °C Survive 95 °C Yellowstone Deep sea vent Heat-stable DNA polymerases for PCR Elongation at 72 °C Survive 95 °C 100 x slower than PolIII. Allow c. 1 min/kb for PCR Taq (Thermus aquaticus) polymerase: no proofreading Pfu (Pyrococcus furiosus) has proofreading 3’>5’ exonuclease, fewer bp changes than Taq but primer shortening reduces specificity. Pfu is more heat stable and more processive than Taq All DNA polymerases require optimal free [Mg2+] DNA and dNTPs bind Mg2+!

1 dsDNA two strands, opposite polarity 2 denaturation separating the DNA strands 3 annealing complementary DNA strands join together to form perfectly matched double-stranded DNA 4 Tm (primer) melting temperature of primer-template complex 5 annealing temperature lowest temperature during PCR cycle 6 DNA primer ca 20 nucleotide single-stranded DNA (synthetic oligo-nucleotide) 7 priming providing partially double-stranded DNA as substrate for DNA polymerase 8 elongation synthesising a complementary DNA strand 9 PCR cycle 95° , 55°, 72° 30 sec to 2 min each

Typical cycling parameters: 30 sec 95°C denaturation of DNA PCR primer specificity PCR can be used to amplify a specific fragments from total genomic DNA if the priming sites are unique, and the annealing conditions are optimal Annealing temperature of primers: Tm (ºC)  4 x (G+C) +2 x (A+T) (there are more complicated formulae but none is perfect) Example: 20 bp 50% G+C: Tm = 60 ºC At Tm, 50% of DNA is annealed, efficient priming possible at higher temperature. Typical cycling parameters: 30 sec 95°C denaturation of DNA 30 sec 55°C annealing of primer 90 sec 72°C elongation (dNTP incorp.)

Optimising PCR specificity Primer design: 3’ end of primer must be specific, preferably A/T G/C G/C AGC3’ 5’ 5’ Tail does not need to anneal Template

PCR (DNA polymerase) inhibitors Bad PCR primers 3’ overlap Hairpin PCR (DNA polymerase) inhibitors present in many impure DNA samples (blood, tissue, food etc) purify DNA or dilute

Increasing the specificity of PCR Hot start PCR: Heat samples to 95 C before activating DNA polymearase Cool to Tm perfectly annealed primers are elongated first Some polymerases are supplied in inactive form, e.g. bound to a specific antibody. Incubation at 95 C removes antibody and activates polymerase. Touch down PCR (in addition to hot start): Start with high annealing temperature (e.g. 65C) Decrease annealing temperature 1C for every cycle Priming starts at highest possible temperature (best specificy) Correct fragment wrong fragments cold start hot start + touch down 1 kb 0.3 kb

usually smaller than correct fragments Increasing PCR specificity using 3’ nested primers correct PCR fragment wrong fragment from unknown sequence usually smaller than correct fragments Re-amplify mixture of correct and wrong PCR fragments 3’ nested primer correct PCR fragment wrong fragments are not amplified because 3’ end of nested primer finds no match specific

PCR contamination How to prevent a single DNA molecule landing in your sample? Serious problem where the same primer pair is used repeatedly DNA is very stable. Soon PCR fragments are erywhere! Disposable gloves Filter tips Synthesize DNA using dUTP instead of dTTP Add heat labile Uracil-DNA Glycosylase to template Normal DNA not affected PCR fragments containing U are destroyed

Model Answers Non-specific bands can be recognized by size, 1-primer PCR, restriction digests, re- amplification using 3’-nested primers, or sequencing. Specificity can be improved by good primers, hot start touchdown PCR, high annealing temperature, low Mg++, enhancing agents (DMSO), not too many cycles. [I assume that you would chose correct amounts of template, primers, dNTPs, DNA polymerase]. PCR fragments are the most common and dangerous sources of template contamination. Clean technique (labcoat, gloves, filter tips, no aerosols (do not empty pipettes completely; avoid DNA dust), laminar flow). Different rooms and labcoats, gloves for PCR setup and amplification. No template controls. dUTP instead of TTP+U-DNA glycohydrolase or similar. Note, autoclaving does not destroy DNA!