Ex No.

- 03 Polymerase Chain Reaction

Aim:To amplify the isolated DNA by PCR. Principles:PCR allows amplification of the target DNA into millions of copies by following repetitive cycling of 3 specific successive steps, each time doubling the DNA molecule. The final number of copies of the DNA can be expressed by the formula. 2nx m Where, n=no. of cycle m=no. of copies of original template PCR amplification involves three major steps which is done in an automated thermal cycler which can heat and cool the tubes with the reaction mixture in a very short time. The temperature and time can be set accordingly to the requirement of each reaction. Steps in PCR are explained as below. 1. Denaturation at 94C:During this step, the double strand melts open to single stranded DNA , all enzymatic reaction stop. 2. Annealing at 54C:During this step Oligonucleotide primer anneals with the single stranded DNA Oligonucleotide constantly try to attach with the complementary bases in the dissociated template DNA by hydrogen bond. The more stable bonds last a little bit longer (primer that fit exactly) and on that little piece of DNA (template & primer), the polymerase can attach and start copying the

template. Once there are a few bases built in the hydrogen bond is so strong between the template and the primers do not break anymore. 3. Extension at 72C:This is the ideal working temperature for the polymerase, where the primers extend in the 3direction with polymerase in the presence of dNTPs. Primers that are on positions with no exact match get loose again (because of the higher temperature) and dont give extension of the fragment. The bases (complementary to the template) are coupled to the primer on the 3side (the polymerase adds dNTPs from 5 to 3 reading the template from 3 to 5side, bases are added complementary to the template. Materials: Template DNA (genomic, plasmid, cosmid, bacterial/yeast colony, etc.) Primers (resuspended to a known concentration with sterile TE) Buffer (usually 10X, usually sold with Taq polymerase or you can make your own) Note: some different buffer receipes follow at the end of this protocol MgCl2 (25mM is convenient) Taq polymerase dNTPs (2mM stock) Note: a 2mM stock of dNTPs means that the final concentration of each dNTP (dATP, dCTP, dGTP, and dTTP) is 2mM -- NOT that all dNTPs together make 2mM. dNTPs come as 100mM stocks -- thaw and add 10L of each dNTP to 460L of ddH20 to make 2mM. Store at -20C.

Reaction Components of PCR:1. DNA polymerase: Most commonly used DNA polymerase is Taq polymerase which is isolated from a bacterium found in hot spring known as Thermus aquaticus. It works optimally at 72C and over pH range 7.0-7.5, adding 100 nucleotides per second to the primer. It is a heat stable enzyme withstanding repeated Denaturation cycles.

2. Deoxynucleotide triphosphates (dNTPs): It is available as a mixture of deoxyadinosine triphosphates (dATP), deoxycytidine triphosphates (dCTP), deoxyguanosine triphosphates (dGTP) and deoxythymidine triphosphates (dTTP). Usually each dNTP is used at concentration between 50m and 200m. these dNTPs attach to the free 3 hydroxyl group of the primer and form a complementary to the template strand. 3. Reaction buffer and MgCl2 The buffer most often used in PCR is 10mM Tris buffer with pH range of 8.5-9.0 at 25C. Because the pH of the Tris buffer decreases by 0.3 units by each 10C rise in the temperature, a buffer made to pH 8.8 at 25C will have a pH of 7.4 at 72C. The dNTPs bind Mg2+ and hence the reaction mixture must contain excess Mg2+. Generally the Mg2+ concentration in the reaction mixture is 0.5 to 2.5mM greater than the concentration of dNTPs. Concentration of Mg2+ also influences the efficiency of primer to template annealing. 4. PCR Primers: Primer concentration should not be greater than 1m. In general, primers should be 20-30 nucleotides in length, which allows reasonably high annealing temperature. The primer should be made with an approximately equal number of each of the 4 bases avoiding regions of unusual sequence such as polypurines, polypyrimidines or repetitive motifs. There should be no 3 end complementarily between primer pairs to avoid primer-dimer formation. 5. Template DNA: Intact template DNA is a prerequisite of successful PCR. Over abundance of template DNA will favor annealing of the 2 strands of template sequence rather than annealing to primer pair and thereby reduce the efficiency of PCR. PCR Protocol:The following reagents were added to the PCR tubes to a final volume of 50l. Use 2l of 60ng/l or 100ng/l DNA

 Use 2l of each primer at 3.2pmole/l concentration or 2.5l of each primer at 100ng/l concentration 5l 10x PCR Buffer w/ Mg 1l 25mM MgCl2 1l dNTP 0.25l Taq 36.75l sterile water to equal a 50l rxn (*if not making a master mix, dilute Taq so that you can add 1l of Taq and 36l sterile water to equal a 50l rxn) 1. Keep the reagents on ice. 2. Add the Taq last, and keep it in the freezer until you are ready to add it. 3. Vortex briefly and quick spin. 4. Cycle: 95C for 1-5minutes (usually 4min) 95C for 1min 55C for 1min 72C for 1.5 to 2min (usually 2min) 72C for 10min 4C hold Cycle 30 times