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The polymerase chain reaction (PCR) is an enzymatic process that allows for the detection of specific genes within an environmental DNA sample. PCR utilizes.
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Laboratory for Environmental Pathogens Research Department of Environmental Sciences University of Toledo
Background information The polymerase chain reaction (PCR) is an enzymatic process that allows for the detection of specific genes within an environmental DNA sample. PCR utilizes short, user defined DNA sequences called oligonucleotide primers, the sequence of which are complementary to target regions of genes known to encode for specific microbial functions (e.g. contaminant degradation). In brief, the DNA sample is denatured to produce single stranded DNA, called template DNA, to which the oligonucleotide primers can bind. The enzyme DNA polymerase then adds nucleotide bases to the end of each primer, using the template DNA as a guide to extend the primer thereby producing new double stranded DNA. This process is repeated for a number of cycles to enrich the DNA sample for the desired genes targeted by the oligonucleotide primers. Since each cycle of PCR involves creating two new double stranded DNAs from each DNA molecule present, the amount of DNA theoretically doubles with every cycle of PCR. Therefore, after two cycles the concentration of DNA increases by 2^2 - fold, after 3 cycles a 2 3
Materials Template DNA (genomic, plasmid, bacterial colony, etc.) Primers (resuspended in sterile water or TE to a concentration of 100 mM ) Buffer (usually 10X , usually sold with Taq polymerase or you can make your own) MgCl 2 (available in 25mM or 50 mM stocks) Bovine serum albumin (BSA, 30 mg ml
- 1 stock) Taq DNA polymerase dNTPs ( 2.5 mM working solution) Note: a 2.5 mM working solution of dNTPs means that the final concentration of each dNTP (dATP, dCTP, dGTP, and dTTP) is 2.5 mM, not that all dNTPs together make 2.5 mM. dNTPs come as 100 mM stocks. Therefore, to make the working solution thaw and add 20 μL of each dNTP to 720 μL of nuclease-free water, mix thoroughly and aliquot in 100 μl volumes. Store at - 20°C. Sterile, nuclease-free water Gloves PCR thermalcycler Pipettes (1- 10 μl, 5- 50 μl, 20- 200 μl, and 100- 1000 μl) and aerosol barrier pipette tips PCR tubes (0.2 ml or 0.5 ml) Master mix tubes (1.5 ml microcentrifuge tubes) PCR allows the production of more than 10 million copies of a target DNA sequence from only a few molecules. Therefore, PCR is very sensitive to contamination from non-target DNA. Several steps should be taken to reduce the chance for contamination, including:
buffers are often available in 10X concentration and are sometimes Taq formulation-specific. Although most protocols recommend a final buffer concentration of 1X, increasing the concentration to 1.5X might result in increased PCR product yield. PCR primers PCR primers are short fragments of single stranded DNA (15-30 nucleotides in length) that are complementary to DNA sequences that flank the target region of interest. The purpose of PCR primers is to provide a “free” 3’-OH group to which the DNA polymerase can add dNTPs. The C and G nucleotides should be distributed uniformly throughout of the primer and comprise approximately 40-60% of the bases. 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. 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. The melting temperature of flanking primers should not differ by more than 5°C. Therefore, the GC content and length must be chosen accordingly. If the primer is shorter than 25 nucleotides, the approx. melting temperature (Tm) is calculated using the following formula: , where G , C , A , and T , are the number of respective nucleotides in the primer. 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. The PCR annealing temperature (TA) should be approximately 5°C lower than the primer melting temperature.
MgCl 2 The concentration of MgCl 2 influences the stringency of the interaction between the primers and the template DNA. The range of MgCl 2 usually tested is from 0.
melting temperature of primer-template DNA duplex. In most cases the TA is between 50 and 65o^ C. However, if nonspecific PCR products are obtained in addition to the expected product, the annealing temperature should be optimized by increasing it stepwise by 1- 2 o C. Extension The processivity of Taq DNA polymerase is approximately 150 nucleotides sec
In general we do not mix the reagents for each individual reaction. Rather, a master mix is made in which enough of each reagent to satisfy all reactions is combined into one tube, and then dispensed among the individual reaction tubes. Template DNA is then added to each tube. This limits pipetting errors as well as the potential for contamination, as the reagent tubes are opened and dispensed from only one time per reaction set-up. Notice also that the number of reactions (“rxn #”, in green) calculated by this spreadsheet is one more than the actual number to be run (six vs. seven). This accounts for any pipetting errors and ensures that enough master mix is made to satisfy all of the reactions. In this example, PCR is being performed on 0.5 μl of DNA (final concentration = 1 ng μl