1. Primer Design: Size Genome Sizes: Prokaryote genome size: –M genitalium = 0.5 million bp; E coli = 4 million bp Eukaryote genome size: –Yeast = 12 million; human = 3 billion; maize = 4 billion bp Primer complexity / size: 4 17 17 179 800 00017-base oligo has “complexity” or number of possible sequences of 4 17 = 17 179 800 000=fold –1/4000 chance of occurring randomly in E coli genome –1/4 chance of occurring randomly in maize genome MAKE PRIMERS LONG ENOUGH FOR TARGET GENOMES

2. Primer Design: Sequence 1 Rules for Primer Design: Rules for Primer Design: (adapted from Innis and Gelfand, 1991): primers should be 17-28 bases in length; base composition should be 50-60% (G+C); primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming; T m s between 55-70 o C are preferred; runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided; primer self-complementarity (ability to form 2 o structures such as hairpins) should be avoided. 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially Primers should not be too degenerate

3. Primer Design: Sequence 2 Genome Sequence Known:

4. Primer Design: Sequence 2 Primer Properties:

5. Primer Design: Degeneracy One makes degenerate primers – or effective mixtures of many primers of similar but non-identical sequence – to amplify DNA that is evolutionarily related to known sequence(s). Eg: for “evolutionary” PCR, or discovery / characterisation of genes in species related to a model organism; For detection / discovery of novel organisms, for example, in environmental investigations such as oceanic / hot spring water bacterial sampling, biofilm diversity etc. Method: multiply align as many related sequences as possible, choose conserved areas of sequence, design primers so as to maximise the probability of amplifying as many sequence relatives as possible Examples: Detection of related viruses in faecal samples; detection of new bacterial species by rDNA gene PCR

6. Primer Design: Degeneracy 2

7. Primer Design: Degeneracy 3

8. Detection Methods: (A)Ethidium bromide- stained agarose gel of PCR products from various grasses infected with mastreviruses (B)Southern blot of agarose gel probed with MSV-specific labelled DNA Note different intensities Note different intensities of bands in (a) and (b): this is due to the sequences being more or less related to the probe sequence

9. Factors Affecting PCR: Chemical:Physical: Primer design Annealing temperature and time Nature and length of template Denaturing temperature and time Reaction buffer Elongation time Detection method Number of cycles

10. Annealing 1: temperatures Usually set at ~5 o C below T m T m Determined by length and GC content of oligo Formula for T m calculation: Melting Temperature T m ( o K)={ΔH/ ΔS + R ln(C)}, Or Melting Temperature T m ( o C) = {ΔH/ ΔS + R ln(C)} - 273.15 where ΔH (kcal/mole) : H is the Enthalpy. Enthalpy is the amount of heat energy possessed by substances. ΔH is the change in Enthalpy. In the above formula the ΔH is obtained by adding up all the di-nucleotide pairs enthalpy values of each nearest neighbor base pair. ΔS (kcal/mole) : S is the amount of disorder a system exhibits is called entropy. ΔS is change in Entropy. Here it is obtained by adding up all the di-nucleotide pairs entropy values of each nearest neighbor base pair. An additional salt correction is added as the Nearest Neighbor parameters were obtained from DNA melting studies conducted in 1M Na+ buffer and this is the default condition used for all calculations. ΔS (salt correction) = ΔS (1M NaCl )+ 0.368 x N x ln([Na+]) Where N is the number of nucleotide pairs in the primer ( primer length -1). [Na+] is salt equivalent in mM. http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html [Na+] calculation: [Na+] = Monovalent ion concentration +4 x free Mg2+. http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

11. Basic T m Calculations The simplest formula is as follows: T m = 4°C x (G +C) + 2°C x (A + T) This formula is valid for oligos <14 bases and assumes that the reaction is carried out in the presence of 50mM monovalent cations. For longer oligos, the formula below is used: T m = 64.9°C + 41°C x (G + C – 16.4)/N Where N is the length of the primer.http://www.promega.com/biomath/calc11.htm Annealing 2: temperatures

12. Actual temperature used best determined EMPIRICALLY Annealing 3: time and temperatures TIME of annealing not important UNLESS primers >50 bases – the process completes in seconds

13. Actual temperature used best determined EMPIRICALLY Annealing 4: time and temperatures TIME of annealing not important UNLESS primers >50 bases – the process completes in seconds Eg: vary annealing temperature across heating block 35 – 50 o C gradient

14. Factors Affecting PCR: Chemical:Physical: Primer design Annealing temperature and time Nature and length of template Denaturing temperature and time Reaction buffer Elongation time Detection method Number of cycles

15. Template molecule could be: Chromosomal dsDNA (bacterial or eukaryote or viral) Plasmid / viral cccDNA (ss or ds) Single-stranded RNA (viral or cellular) Double-stranded RNA (viral) Template could be: Short: 100 – 500 bases (detection purposes) Medium length: 1000 – 5000 bases (whole gene / whole viral genome amplification) Long: 10 000 – 40 000 bases (mapping cell genomes / whole viral genome amplifications) Nature & Length of Template

16. Effect on denaturation temperature: Need to denature hot enough for long enoughNeed to denature hot enough for long enough –Short templates denature quicker than long –DNA:DNA < DNA:RNA < RNA:RNA in melting temperature –T m depends upon G+C content Usable denaturation temperatures are 88 - 95 o CTime: DNA denatures in just a few seconds at the “strand separation temperature” – BUT as there may be a lag time due to tube material insulating the reactants, so: –around 30 sec at chosen temperature (88 – 95 o C) is required T 1/2 of Taq pol is: –>2 hrs at 92.5 o C –40 minutes at 95 o C –5 minutes at 97.5 o C Nature & Length of Template 2:

17. Factors Affecting PCR: Chemical:Physical: Primer design Annealing temperature and time Nature and length of template Denaturing temperature and time Reaction buffer Elongation time Detection method Number of cycles

18. Elongation Time Elongation time Elongation time (assuming temperature = 70 o C) depends on: LENGTH of target sequence: – Taq pol makes 2 kb in 60 sec STAGE of reaction: – As reactants get reduced with high template concentration, longer times are required – As Taq pol activity falls, may need more time

19. Factors Affecting PCR: Chemical:Physical: Primer design Annealing temperature and time Nature and length of template Denaturing temperature and time Reaction buffer Elongation time Detection method Number of cycles

20. Reaction Buffer 1 concentrated kit component This is almost always provided as a concentrated kit component (to dilute 1/10). It generally contains: NB: reverse transcriptase buffer works well also – 10 – 50 mM Tris-Cl pH 8.3: NB: reverse transcriptase buffer works well also – <50 mM KCl – ~1.5 mM MgCl 2 – *Gelatin or BSA to 100 μM – *Non-ionic detergent 0.01 – 0.1 % v/v * = or other dispersant / protein stabiliser

21. Reaction Buffer 2 0.5 – 2.5 mM > [dNTP]is all chelated Mg 2+ concentration should be 0.5 – 2.5 mM > [dNTP] as otherwise is all chelated TITRATE Mg 2+ !!! Mg 2+ concentration affects: How: Primer annealingincreases probability T m of templateincreases Primer / product association makes more stable Product specificitydecreases Enzyme activityenables / stabilises

22. Reaction Buffer 3 Reaction components:Reason: 0.2 – 1 μM each primerincreases specificity 50 - 200 μM each dNTPincreases specificity 1 Unit Taq polincreases specificity HELIX DESTABILISERS HELIX DESTABILISERS may be needed for HIGH G+C TEMPLATES: DMSO IS BEST Urea, formamide, dimethylformamide (DMF) or dimethylsulphoxide (DMSO) or glycerol all help; DMSO IS BEST Additives also help for LONG TEMPLATES Polyethylene glycol (PEG) can help when [template] is LOW.

23. Factors Affecting PCR: Chemical:Physical: Primer design Annealing temperature and time Nature and length of template Denaturing temperature and time Reaction buffer Elongation time Detection method Number of cycles

24. How Many Cycles is Enough? 30 cycles = 10 9 -fold amplification 40 cycles = 10 12 -fold amplification 50 cycles – 10 15 -fold amplification….?? 50 cycles – 10 15 -fold amplification….?? Innis & Gelfand, 1990 50 copies of target DNA: 40 – 45 cycles 5 x 10 5 copies of target: 25 – 30 cycles

25. Plateau Effect in DNA Amplification: Log 2 [product] Cycle number 10 20 30 4010 20 30 40 50 Theoretical Actual

26. How to Amplify More DNA? Re-amplify reaction product in separate reaction with same primers (just add more reagents): – Advantage – Advantage: further amplification, no reactant depletion – Disadvantage – Disadvantage: further amplification of non- specific products Re-amplify reaction product with DIFFERENT primers (nested primer PCR) after clean-up: – Advantage – Advantage: further amplification WITHOUT same non-specific products – Disadvantage – Disadvantage: more manipulation

27. 1. 30 cycle PCR with Primer Set 1: Nested Primer PCR: ~10 9 -fold amplification 2. 30 cycle PCR with Primer Set 2: ~10 9 -fold amplification ~10 18 Final theoretical amplication: ~10 18 -fold 1 ng…1 gram?? Eg: 1 molecule of 1 kb -> 1 ng (30 cycles) -> …1 gram??

28. How much can you amplify? Theoretically: Consider 100ul reaction with 1uM each primer, 200uM each dNTP, 1000 bp target. Total possible conversion limited by [dNTP] = 0.8 uM assuming A=G=C=T 51.2 ug = 51.2 ug [(0.8 x 640 000 x 10 -6 )/10 000] Practically: only a few micrograms as this reaction cannot go to completion as [reactants] primer + dNTPDNA

29. Amount amplified depends upon [template]: [Product] saturates for [template] above certain limits ONLY RE-AMPLIFY SMALL ALIQUOT OF 1 st REACTION!! C. Soin‚, S. K. Watson, E. P. Rybicki, B. Lucio, R. M. Nordgren, C. R. Parrish, and K. A. Schat. Determination of the detection limit of the polymerase chain reaction for chicken infectious anemia virus. Avian.Dis. 37 (2):467-476, 1993.