The Polymerase Chain Reaction

1. Adapted from a presentation written for Principles of Gene Manipulation · November 6, 2000 Jennifer Cooper · America Madrigal · Laleña Vellanoweth The Polymerase Chain Reaction AMPLIFICATION:

2. Definition of PCR Requirements for PCR PCR Process A. Denaturation B. Annealing C. Extension D. Cycling (repeat A-C) PCR for HLA DQ-alpha 11.06.00 2 AMPLIFICATION:PCR and Its Applications

3. The Polymerase Chain Reaction (PCR) is an invitro method to amplify a specific region of DNA. PCR is extremely sensitive, with the capability of amplifying minuscule quantities of DNA. 11.06.00 3 PCRWhat is it?

4. In the reaction Sample – template Primers High temperature resistant polymerase; e.g., Taq Deoxynucleotide triphosphates – dNTPs (dATP, dGTP, dCTP, dTTP) Buffer Mg++, KCl Thermocycler – instrument programmed to change samples rapidly from one set temperature to another 11.06.00 4 PCRREQUIREMENTS

5. There are three basic steps in PCR 1. Denaturation (~95oC) 2. Annealing (~55oC, but varies) 3. Extension (~72oC) Cycling repeats Steps 1-3 up to 35 times. 11.06.00 5 PCRMETHOD

6. 11.06.00 6 PCRMETHOD – DENATURATION STAGE High temperature separates the two strands. (reference: library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)

7. 11.06.00 7 PCRMETHOD – ANNEALING STAGE Primer length is usually ~20 nucleotides. (reference: library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)

8. 11.06.00 8 PCRMETHOD – EXTENSION STAGE Thermostable polymerase adds dNTPs one at a time at this stage. (reference: library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)

9. 11.06.00 9 PCRMETHOD - CYCLING The average number of cycles = 30 for efficiency reasons. 230 = 1.07 X 109 copies (reference: library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)

10. 11.06.00 10 PCRMETHOD - CYCLING DNA Cycle 1 2 3 4 5 6 … Products 2 4 8 16 32 … After the first two cycles, fragments with the correct length begin to be amplified. (dictated by the placement of the primers)

11. PCRfor HLA DQ-alpha 11.06.00 11 • Biotinylated primers amplify a region of ~250 bp. • Sequence of primers is common to all individuals. • Sequence between primers is polymorphic – may differ between chromosomes and among individuals. • PCR picks out the 250 bp HLA DQ- α region from 3.3 X 109 bp of DNA present in 23 chromosomes. • Note that in the kit we are using there are actually six pairs of biotinylated primers (12 primers total). • Only 1 pair will amplify the HLA DQ- α region • The other five pairs of primers amplify genes that we are not interested in for this class. The analysis for these genes employs a different probe strip than the HLA DQ- α probe strip that we will be using. • After agarose gel electrophoresis six bands will be seen, only one of which is the band of interest for this class.

12. The human male karyotype: 22 homologous chromosomes + X and Y

14. PCRfor HLA DQ-alpha • Products of PCR reaction • >106 double strand fragments ~ 250 bp in length. • All are biotinylated (biotinylated primers). • 1/2 = DQ-α allele on one copy of your Chromosome 6; 1/2 = DQ-α allele on the other copy.

15. 11.06.00 15 PCRREFERENCES 1. Gene Cloning: an introduction. T.A. Brown 2. The world wide web: http://sunsite.berkeley.edu/PCR/whatisPCR.html http://www.accessexcellence.org/AB/GG/polymerase.html http://www.sciam.com/1998/0598issue/0598working.html http://www.faseb.org/opar/bloodsupply/pcr.html http://faculty.plattsburgh.edu/donald.slish/PCR.html http://library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html

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17. PCR RequirementsThe Details

18. Amount Needed very small; intact DNA from one cell to see on a gel, need 1011 final copies; need 104 starting copies Concern –competition with primers for annealing by Too much starting template Too much product from excessive cycling Remember, the association rate of two strands increases with the square root of the length of the DNA. Longer strands anneal more quickly than shorter. So . . . Templates and products are longer than primers. In high concentration, they reanneal before primers can anneal. 11.06.00 4 PCRREQUIREMENTS: sample - template

19. Even degraded DNA is OK if sample is large enough Fossils Remains Old samples from crime scenes PCRREQUIREMENTS: sample - template

20. Two primers of known sequence flank region you are interested in anneal to opposite strands of template prime toward the region between them non-complementary to each other lack internal complementarity of sufficient length to anneal to unique site in the genome (~20 nt) 1/420 = 1 site of identical sequence/1 X 1012 bp chance that any one of the 4 bases will be at a given site = 1/4 have similar annealing temperatures present in excess (0.1 – 1.0 uM each) favor annealing of primer over reannealing of strands sufficient for amplification through 25-30 cycles PCRREQUIREMENTS - primers

21. must be present in sufficient excess to complete extension through all cycles (~200 M in each dNTP) must not be present in such high excess that Mg++ ions are complexed and unavailable as cofactors for polymerase activity PCRREQUIREMENTS - dNTPs

22. The buffer, e.g., Tris base adjusted to a specific pH with HCl maintains pH pH 8.3 is optimum for Taq. pH optimum keeps protein folded in a conformation at which it is enzymatically active. Different temperature-insensitive polymerases have different pH optima PCRREQUIREMENTS - “buffer” components

23. Monovalent salt, e.g., KCl to contribute to correct folding of enzyme and thereby to contribute to optimum activity of enzyme PCRREQUIREMENTS - “buffer” components

24. Mg++ (MgCl2, MgSO4) Mg++ is a cofactor for DNA polymerases for Taq, required free [Mg++] = ~2mM calculated [Mg++] may differ from the actual free [Mg++] Positively charged Mg++ is complexed by ionic bonding with the negative charges on primers, template, and dNTPs Mg++ must be uncomplexed (free) to act as a cofactor for the polymerase PCRREQUIREMENTS - “buffer” components

25. Mg++ (cont’d) Determining the optimal concentration of Mg++ is the most important step in setting up PCR conditions too little - polymerase can’t work too much - favors annealing of primers to mismatched locations on the template PCRREQUIREMENTS - “buffer” components

26. 1-2 units of enzyme/100 l reaction High temperature resistant able to remain active through up to 35 cycles with DNA denaturation at 95oC Example: Taq polymerase isolated from Thermophilus aquaticus PCRREQUIREMENTS - polymerase

27. Many different polymerases available some have both polymerase and editing (exonuclease) activities PCRREQUIREMENTS - polymerase (cont’d) Pfu polymerase can edit. Taq polymerase can not. Taq polymerase is more likely to misincorporate a dNTP.

28. Many different polymerases available leave different types of ends blunt single A 3’overhang each isolated from a different organism which has evolved to survive at high temperatures deep water vents hot springs PCRREQUIREMENTS - polymerase (cont’d)

29. Denaturation temperature high enough to overcome attractive energy of H-bonds between bases of the complementary template and product strands 95oC provides sufficient energy to separate even long strands PCRREQUIREMENTS - denaturation of dsDNA

30. The lower the temperature, the easier it is for 2 strands of DNA to pair with each other So the chosen annealing temperature must be High enough to prevent hybridization of primers to imperfectly complementary template sequences (i.e., non-specific annealing) Not so high that the primers can’t anneal to template DNA at all PCRREQUIREMENTS - annealing

31. What determines the optimum annealing temp length of primer - the longer, the higher the optimum annealing temp will be longer the primer, the more H-bonds So, the more H-bonds, more likely 2 strands are to anneal or stay annealed % GC - the more GC, the higher the optimum annealing temp will be GC base pairs have 3 H-bonds; AT base pairs have only 2 So, the more GC, more likely 2 strands are to anneal or stay annealed the  the [salt], the  the optimum annealing temp will be positive ions in salt are counterions to the negatively charged sugar-phosphate backbone of the ds DNA positive counterions prevent repulsive forces of negative charges from pushing the strands apart PCRREQUIREMENTS - annealing

32. Annealing temperature for PCR is often set at 5oC below the Tm Tm = temperature at which 50% of the possible correct primer/template complexes are unformed Estimate Tm for primers 10-23 nt long in 1M salt Tm (oC) = 4 (G+C) + 2 (A+T) PCRREQUIREMENTS - annealing

33. Way to minimize early non-specific annealing that causes primer dimers amplification of incorrect product Hot start Why might early annealing be expected to be non-specific?

34. Enzyme is not mixed with reaction until sample has reached denaturation temperature manual addition of enzyme at 95C polymerase separated from other reagents by layer of solid wax wax melts at denaturation temperature, polymerase mixes with reagents, wax rises to top and prevents evaporation start with antibody/polymerase complex antibody denatured and releases enzyme at 95C Hot start

35. Note: the longer the expected product, the longer the extension time required exact time depends on rate of progression of the specific polymerase Extension temperature optimal temperature for enzyme determined for each enzyme empirically usually around 72oC PCRREQUIREMENTS - extension

36. Thermocycler - instrument programmed to change samples rapidly from one set temperature to another PCRREQUIREMENTS

37. Way to prevent evaporation of water from reaction at high temperatures Why? evaporation raises concentrations of reaction solutes  inhibition of reaction How? Thermocycler applies heat to the top of the reaction tube, thereby preventing condensation or, overlayer the reaction with mineral oil, preventing evaporation PCRREQUIREMENTS

38. Way to heat and cool the sample Solid heating/cooling block that holds samples Efficient conduction of heat between heating/cooling block and sample pressure applied from top pushes walls of tube directly against block, eliminating air space, or mineral oil is used to fill in air space between heating block and sample PCRREQUIREMENTS - thermocycler

39. No template control should be no product if there is, contaminating DNA is present Known positive (if possible) should be a product tells you all reaction components are working may tell you what your product should look like PCRREQUIREMENTS - controls

40. Size markers show what size your product is and if you know what size to expect, whether you are getting the expected product Results usually analyzed by gel electrophoresis PCRREQUIREMENTS - controls (cont’d)

41. Contamination of pipettors Use aerosol barrier pipet tips Contamination of supplies and reagents UV irradiation base/acid treatment of reusable supplies PCRREQUIREMENTS -Minimizing Contamination

42. Contamination of work area with sample or product Perform steps at separated benches or rooms sample prep reaction set up thermocycling product analysis Prevent aerosols containing PCR products centrifuge reagents and products before opening tube also prevents contamination of reagents from gloves uncap tubes carefully PCRREQUIREMENTS -Minimizing Contamination (cont’d)

43. Contamination of reaction mix Use aerosol barrier pipettor tips Use distilled deionized water Add DNA to the reaction last PCRREQUIREMENTS Minimizing Contamination (cont’d)

44. No yield Extra or incorrect products Primer dimers Misincorporation PCRREQUIREMENTS -Troubleshooting

45. No yield Were all reagents included? Insufficient denaturation? Higher temp Check conditions for transfer of heat from block to tube Active nucleases or proteases present in rx? Insufficient free Mg++? Bad primers? Degraded Wrong sequence PCRREQUIREMENTS -Troubleshooting

46. Extra or incorrect products Mispriming annealing temp too low may need Hot Start too much Mg++; facilitates misannealing primer sequence insufficiently specific [dNTP] too high Too much polymerase Annealing and/or extension time too long Too many cycles; rare misprimed products become amplified Template contamination PCRREQUIREMENTS -Troubleshooting

47. Primer dimers What are they? 5’---------------------AT3’ 3’TA----------------------5’ Visible below the 100 bp marker on gel Can appear even when 3’ ends are not complementary PCRREQUIREMENTS -Troubleshooting

48. Primer dimers Causes Primer excess too great 3’ primer complementarity Insufficient target template Too many cycles Annealing temperature too low Hot start may be required to avoid initial primer/primer annealing. Primers insufficiently specific (too short) PCRREQUIREMENTS -Troubleshooting

49. Misincorporation Excess of dNTPs too great dNTPs present in unequal concentrations; one is exhausted before others Polymerase lacking exonuclease (editing) activity Polymerase concentration too high Extension temperature too low [Mg++] too high PCRREQUIREMENTS -Troubleshooting

50. Lane # 1: no template. 2: +  DNA. 3-5: 100X  DNA, [Mg++] 6,8: hair root lysate 7,9: hair shaft lysate Rx in lanes 1-5 were performed with  primers. Rx in lanes 6-9 were performed with HLA-DQ alpha primers. Effects of [template] and [Mg++] on PCR products