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

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

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

PCR REQUIREMENTS In the reaction 11.06.00 11.06.00 4 PCR REQUIREMENTS 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

PCR METHOD There are three basic steps in PCR 1. Denaturation (~95oC) 11.06.00 11.06.00 5 PCR METHOD 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.

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

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

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

11.06.00 11.06.00 9 PCR METHOD - 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)

(dictated by the placement of the primers) 11.06.00 11.06.00 10 PCR METHOD - 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)

PCR for HLA DQ-alpha Biotinylated primers amplify a region of ~250 bp. 11.06.00 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.

The human male karyotype: 22 homologous chromosomes + X and Y 11.06.00 The human male karyotype: 22 homologous chromosomes + X and Y

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PCR for HLA DQ-alpha Products of PCR reaction 11.06.00 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.

PCR REFERENCES 1. Gene Cloning: an introduction. T.A. Brown 11.06.00 11.06.00 15 PCR REFERENCES 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

11.06.00 After a long process, this PowerPoint presentation has now ended. We hope it is of good help in your studies.

PCR Requirements The Details 11.06.00 PCR Requirements The Details

PCR REQUIREMENTS: sample - template 11.06.00 11.06.00 4 PCR REQUIREMENTS: sample - template 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.

PCR REQUIREMENTS: sample - template 11.06.00 PCR REQUIREMENTS: sample - template Even degraded DNA is OK if sample is large enough Fossils Remains Old samples from crime scenes

PCR REQUIREMENTS - primers 11.06.00 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

PCR REQUIREMENTS - dNTPs 11.06.00 PCR REQUIREMENTS - dNTPs 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

PCR REQUIREMENTS - “buffer” components 11.06.00 PCR REQUIREMENTS - “buffer” components 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

PCR REQUIREMENTS - “buffer” components 11.06.00 PCR REQUIREMENTS - “buffer” components Monovalent salt, e.g., KCl to contribute to correct folding of enzyme and thereby to contribute to optimum activity of enzyme

PCR REQUIREMENTS - “buffer” components 11.06.00 PCR REQUIREMENTS - “buffer” components 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

PCR REQUIREMENTS - “buffer” components 11.06.00 PCR REQUIREMENTS - “buffer” components 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

PCR REQUIREMENTS - polymerase 11.06.00 PCR REQUIREMENTS - polymerase 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

PCR REQUIREMENTS - polymerase (cont’d) 11.06.00 PCR REQUIREMENTS - polymerase (cont’d) Many different polymerases available some have both polymerase and editing (exonuclease) activities Pfu polymerase can edit. Taq polymerase can not. Taq polymerase is more likely to misincorporate a dNTP.

PCR REQUIREMENTS - polymerase (cont’d) 11.06.00 PCR REQUIREMENTS - polymerase (cont’d) 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

PCR REQUIREMENTS - denaturation of dsDNA 11.06.00 PCR REQUIREMENTS - denaturation of dsDNA 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

PCR REQUIREMENTS - annealing 11.06.00 PCR REQUIREMENTS - annealing 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

PCR REQUIREMENTS - annealing 11.06.00 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

PCR REQUIREMENTS - annealing 11.06.00 PCR REQUIREMENTS - annealing 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)

Why might early annealing be expected to be non-specific? 11.06.00 Hot start Way to minimize early non-specific annealing that causes primer dimers amplification of incorrect product Why might early annealing be expected to be non-specific?

11.06.00 Hot start 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

PCR REQUIREMENTS - extension 11.06.00 PCR REQUIREMENTS - extension 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

11.06.00 PCR REQUIREMENTS Thermocycler - instrument programmed to change samples rapidly from one set temperature to another

11.06.00 PCR REQUIREMENTS 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

PCR REQUIREMENTS - thermocycler 11.06.00 PCR REQUIREMENTS - thermocycler 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

PCR REQUIREMENTS - controls 11.06.00 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

PCR REQUIREMENTS - controls (cont’d) 11.06.00 PCR REQUIREMENTS - controls (cont’d) 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

PCR REQUIREMENTS - Minimizing Contamination 11.06.00 PCR REQUIREMENTS - Minimizing Contamination Contamination of pipettors Use aerosol barrier pipet tips Contamination of supplies and reagents UV irradiation base/acid treatment of reusable supplies

PCR REQUIREMENTS - Minimizing Contamination (cont’d) 11.06.00 PCR REQUIREMENTS - Minimizing Contamination (cont’d) 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

PCR REQUIREMENTS Minimizing Contamination (cont’d) 11.06.00 PCR REQUIREMENTS Minimizing Contamination (cont’d) Contamination of reaction mix Use aerosol barrier pipettor tips Use distilled deionized water Add DNA to the reaction last

PCR REQUIREMENTS - Troubleshooting 11.06.00 PCR REQUIREMENTS - Troubleshooting No yield Extra or incorrect products Primer dimers Misincorporation

PCR REQUIREMENTS -Troubleshooting 11.06.00 PCR REQUIREMENTS -Troubleshooting 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

PCR REQUIREMENTS -Troubleshooting 11.06.00 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

PCR REQUIREMENTS -Troubleshooting 11.06.00 PCR REQUIREMENTS -Troubleshooting 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

PCR REQUIREMENTS - Troubleshooting 11.06.00 PCR REQUIREMENTS - Troubleshooting 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)

PCR REQUIREMENTS - Troubleshooting 11.06.00 PCR REQUIREMENTS - Troubleshooting 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

Effects of [template] and [Mg++] 11.06.00 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