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Polymerase Chain Reaction (PCR) (The following is modified from http://www.fermentas.com/techinfo/pcr/dnaamplprotocol.htm) PCR allows the production of more than 10 million copies of a target DNA sequence from only a few molecules. The sensitivity of this technique means that the sample should not be contaminated with any other DNA or previously amplified products (amplicons) that may reside in the laboratory environment. DNA sample preparation, reaction mixture assemblage and the PCR process, in addition to the subsequent reaction product analysis, should be performed in separate areas. Fresh gloves should be worn for DNA purification and each reaction set-up. The use of dedicated vessels and positive displacement pipettes or tips with aerosol filters for both DNA sample and reaction mixture preparation, is strongly recommended. The reagents for PCR should be prepared separately and used solely for this purpose. Autoclaving of all solutions, except dNTPs, primers and Taq DNA Polymerase is recommended. Solutions should be aliquotted in small portions and stored in designated PCR areas. Aliquots should be stored separately from other DNA samples. A control reaction, omitting template DNA, should always be performed, to confirm the absence of contamination. Components of the Reaction Mixture Template DNA Usually the amount of template DNA is in the range of 0.01-1 ng for plasmid or phage DNA and 0.1-1 µg for genomic DNA, for a total reaction mixture of 50 µl. Higher amounts of template DNA usually increase the yield of nonspecific PCR products, but if the fidelity of synthesis is crucial, maximal allowable template DNA quantities together with a limited number of PCR cycles should be used to increase the percentage of "correct" PCR products. Nearly all routine methods are suitable for template DNA purification. Although even trace amounts of agents used in DNA purification procedures (phenol, EDTA, Proteinase K, etc.) strongly inhibit Taq DNA Polymerase, ethanol precipitation of DNA and repetitive treatments of DNA pellets with 70% ethanol is usually effective in removing traces of contaminants from the DNA sample. Primers Guidelines for primer selection: PCR primers are usually 15-30 nucleotides in length. Longer primers provide higher specificity. The GC content should be 40-60%. The C and G nucleotides should be distributed uniformly throughout of the primer. More than three G or C nucleotides at the 3' -end of the primer should be avoided, as nonspecific priming may occur. The primer should not be self-complementary or complementary to any other primer in the reaction mixture, in order to avoid primer-dimer and hairpin formation. The melting temperature of flanking primers should not differ by more than 5°C, so the GC content and length must be chosen accordingly. All possible sites of complementarity between primers and the template DNA should be noted. If primers are degenerate, at least 3 conservative nucleotides must be located at the primer's 3'-end. Estimation of the melting and annealing temperatures of primer: If the primer is shorter than 25 nucleotides, the approx. melting temperature (Tm) is calculated using the following formula: Tm= 4 (G + C) + 2 (A + T) :G, C, A, T= number of respective nucleotides in the primer. Annealing temperature should be approx. 5°C lower than the melting temperature. If the primer is longer than 25 nucleotides, the melting temperature should be calculated using specialized computer programs where the interactions of adjacent bases, the influence of salt concentration, etc. are evaluated. dNTP (deoxynucleotide triphosphates = A, T, G, C) The concentration of each dNTP in the reaction mixture is usually 200 µM. It is very important to have equal concentrations of each dNTP (dATP, dCTP, dGTP, dTTP), as inaccuracy in the concentration of even a single dNTP dramatically increases the misincorporation level. Taq DNA Polymerase. Usually 1-1.5 u of Taq DNA Polymerase are used in 50 µl of reaction mix. Higher Taq DNA Polymerase concentrations may cause synthesis of nonspecific products. However, if inhibitors are present in the reaction mix (e.g., if the template DNA used is not highly purified), higher amounts of Taq DNA Polymerase (2-3 u) may be necessary to obtain a better yield of amplification products. Preparation of Reaction Mixture To perform several parallel reactions, prepare a master mix containing water, buffer, dNTPs, primers and Taq DNA Polymerase in a single tube, which can then be aliquoted into individual tubes. Template DNA solutions are then added. This method of setting reactions minimizes the possibility of pipetting errors and saves time by reducing the number of reagent transfers. Reaction Mixture Set Up 1. Gently vortex and briefly centrifuge all solutions after thawing. 2. Put PCR tubes on ice. Typically you should have a positive control and a negative control in addition to other templates to amplify. A negative control has no DNA template, and the positive control using a template and primers known to work in PCR. Reagent Sterile deionized water 10X Taq buffer 2 mM dNTP mix Primer I* Primer II* Taq DNA Polymerase Template DNA Final concentration - 1X 0.2 mM of each 0.1-1 µM 0.1-1 µM 1.25 u / 50 µl The template DNA will be different in tube 1 and tube 10pg-1 µg 3. There should be no template in tube 2. Quantity, for 50 µl of reaction mixture Variable to make 50 µl total volume 5 µl 5 µl 1 µl 1 µl 1 µl a) _____µl for amplification of gene of interest or positive control b) Leave out completely for negative control and increase dH2O by this volume 3. Gently vortex the sample and briefly centrifuge to collect all drops from walls of tube. 4. Place samples in a thermocycler and start PCR. See below for thermocycling temperatures and times. PCR works like DNA replication. DNA is denatured (comes apart into two strands) and short pieces of complementary DNA that flank either end of the gene o f interest anneal to the template DNA at a temperature that is just below their denaturing temperature. This annealing temperature depends on the nucleotide composition of the primer and its complementary DNA. This is because nucleotides G and T have three hydrogen bonds, whereas A and T only have two. The more Gs and Ts you have in your sequence, the higher the annealing temperature will be because of these triple hydrogen bonds. After the primers have annealed to the template DNA, this signals a location for DNA polymerase to attach to the DNA. There must be a short primer next to a single stranded piece of DNA for DNA polymerase to attach. DNA polymerase then utilizes nucleotide triphosphates (A,T,G, and C; also called dNTPs for deoxy-nucleotide triphosphates) to construct the compliment of the template strand. A special form of DNA polymerase from thermophilic bacteria is used in PCR because it continues to work at the high temperatures encountered each time the new strands are denatured (95 oC). Cycling Conditions Amplification parameters depend greatly on the template, primers and amplification apparatus used. Initial Denaturation Step. The complete denaturation of the DNA template at the start of the PCR reaction is of key importance. Incomplete denaturation of DNA results in the inefficient utilization of template in the first amplification cycle and in a poor yield of PCR product. The initial denaturation should be performed over an interval of 1-3 min at 95°C if the GC content is 50% or less. This interval should be extended up to 10 min for GC-rich templates. If the initial denaturation is no longer than 3 min at 95°C, Taq DNA Polymerase can be added into the initial reaction mixture. If longer initial denaturation or a higher temperature is necessary, Taq DNA Polymerase should be added only after the initial denaturation, as the stability of the enzyme dramatically decreases at temperatures over 95°C. Denaturation Step. Usually denaturation for 0.5-2 min at 94-95°C is sufficient, since the PCR product synthesized in the first amplification cycle is significantly shorter than the template DNA and is completely denatured under these conditions. If the amplified DNA has a ve ry high GC content, denaturation time may be increased up to 3-4 min. Alternatively, additives facilitating DNA denaturation - glycerol (up to 10-15vol.%), DMSO (up to 10%) or formamide (up to 5%) - should be used. In the presence of such additives, the annealing temperature should be adjusted experimentally, since the melting temperature of the primer template DNA duplex decreases significantly when these additives are used. The amount of enzyme in the reaction mix should be increased since DMSO and formamide, at the suggested concentrations, inhibit Taq DNA Polymerase by approx. 50%. Alternatively, a common way to decrease the melting temperature of the PCR product is to substitute dGTP with 7-deaza-dGTP in the reaction mix. Primer Annealing Step. Usually the optimal annealing temperature is 5°C lower than the melting temperature of primer-template DNA duplex. Incubation for 0.5-2 min is usually sufficient. However, if nonspecific PCR products are obtained in addition to the expected product, the annealin g temperature should be optimized by increasing it stepwise by 1-2°C. Extending Step. Usually the extending step is performed at 70-75°C. The rate of DNA synthesis by Taq DNA Polymerase is highest at this temperature. Recommended extending time is 1 min for the synthesis of PCR fragments up to 2 kb. When larger DNA fragments are amplified, the extending time is usually increased by 1 min for each 1000 bp. Number of Cycles. The number of PCR cycles depends on the amount of template DNA in the reaction m ix and on the expected yield of the PCR product. For less than 10 copies of template DNA, 40 cycles should be performed. If the initial quantity of template DNA is higher, 25 -35 cycles are usually sufficient. Final Extending Step. After the last cycle, the samples are usually incubated at 72°C for 5-15 min to fill-in the protruding ends of newly synthesized PCR products. Also, during this step, the terminal transferase activity of Taq DNA Polymerase adds extra A nucleotides to the 3'-ends of PCR products. Therefore, if PCR fragments are to be cloned into T/A vectors, this step can be prolonged to up to 30 min. An example of a PCR cycling program is shown below: Initial DNA denaturing 95 o for 5 min DNA denaturing 95 o for 30s Primer annealing 54 o for 30 s Extension 75 o for 30 s Repeat this for 30 cycles Final extension 75 o for 5 min Final temperature hold at 4 o until you can put your samples in the freezer PCR methods There are a variety of variations on this basic PCR technique. These variations are used when a particular template, or situation exists, for which basic PCR is less efficient. 1. Hot-start PCR - to reduce non-specific amplification. Can also be done by separating the DNA mixtures from enzyme by a layer of wax which melts when heated in cycling reaction. 2. "Touch-down" PCR - start at high annealing temperature, then decrease annealing temperature in steps to reduce non-specific PCR product. Can also be used to determine DNA sequence of known protein sequence. 3. Nested PCR - use to synthesize more reliable product - PCR using a outer set of primers and the product of this PCR is used for further PCR reaction using an inner set of primers. 4. RT-PCR (reverse transcriptase) - using RNA-directed DNA polymerase to synthesize cDNAs which is then used for PCR and is extremely sensitive for detecting the expression of a specific sequence in a tissue or cells. It may also be use to quantify mRNA transcripts. This is also called Quantiative RT-PCR, Competitive Quantitative RT-PCR, RT in situ PCR, Nested RT-PCR. 5. RACE (rapid amplificaton of cDNA ends) - used where information about DNA/protein sequence is limited. Amplify 3' or 5' ends of cDNAs generating fragments of cDNA with only one specific primer each (+ one adaptor primer). Overlapping RACE products can then be combined to produce full cDNA.. 6. DD-PCR (differential display) - used to identify differentially expressed genes in different tissues. First step involves RT-PCR, then amplification using short, intentionally nonspecific primers. Get series of band in a high-resolution gel and compare to that from other tissues, any bands unique to single samples are considered to be differentially expressed. 7. Multiplex-PCR - 2 or more unique targets of DNA sequences in the same specimen are amplified simultaneously. One can be use as control to verify the integrity of PCR. Can be used for mutational analysis and identification of pathogens. 8. Q/C-PCR (Quantitative comparative) - uses an internal control DNA sequence (but of different size) which compete with the target DNA (competitive PCR) for th e same set of primers. Used to determine the amount of target template in the reaction.