PCR amplification of 16S rRNA gene

1. PCR amplification of 16S rRNA gene Ros. Brown School of Biology Newcastle University UK

2. 16S rRNA gene The sequence of the 16S rRNA gene has been widely used as a means to identify an unknown bacterium to the genus or species level. The Polymerase Chain Reaction (PCR) is a technique that 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.

3. PCR reagents and conditions Taq DNA polymerase: a thermostable enzyme of approximately 94 kDa isolated in 1976 from Thermus aquaticus strain YT-I(1), This unmodified enzyme replicates DNA at 72°C. The enzyme catalyzes the polymerization of nucleotides into duplex DNA in the 5' to 3' direction in the presence of magnesium ions and possesses a 5' to 3' exonuclease activity. Buffer: Most buffers used in PCR are based on Tris-KCI or Tris-Ammonium Sulphate at pH 8.3-8.8. MgCl2: Most DNA polymerases require an optimum of 1.5-2.5 mM Mg+ usually supplied in the form of MgCl2 or MgS04. dNTPs: are supplied at concentrations around 200-400 mM (50mM each), but the concentration can be much higher in the case of long-PCR to generate very long products. Template: Theoretically a single DNA molecule can be picked by the enzyme but the amounts usually used are from nanograms to micro grams of DNA. Primers: Usually used at concentrations around 400-500 nM, however the concentration of degenerate primers can be up to 10 fold higher to compensate for the reduced concentration of the specific oligomer.

4. PCR amplification PCR amplification cycles consist of 3 main steps: a. Denaturing step leading to strand separation which occurs at high temperature (usually over 80°C and typically 94-95 oC). The denaturing step usually lasts for 15 to 30 sec but can be extended for long fragments b. Annealing step corresponds to primer hybridization with the template. The annealing temperature is dependent on the primer sequence and base composition and usually is kept lower than 72°C. Annealing time of 30-45 seconds commonly used. Increasing annealing time does not appreciable influence the outcome of PCR reactions-polymerase has reduced activity between 45-65°C, therefore longer annealing times may increase the likelihood of unspecific amplification. Tm of primer in oC = (no. of GC x 4) + (no. AT x 2) Ideally the Tm of each primer (forward and reverse) should be similar The annealing temperature is usually (Tm – 50C) c. Extension step is normally at 72°C for about 1 min for every 1 kb to be amplified (depends on the DNA polymerase used). Usually a final extension at 72°C for 5 to 10 min is included at the end of the cycle to allow the completion of the extension of all the products.

5. Controls It is vital to run both positive and negative PCR controls in parallel with your samples. Negative controls usually test for contaminating DNA that might be introduced during sample preparation from the environment or via contaminated reagents. The template is omitted from the negative controls which must contain all the other ingredients of the reaction. Given the sensitivity of the technique (in theory a single DNA molecule can be detected), it is necessary to observe strict rules in terms of wearing gloves, using autoclaved solutions and working in a clean area designated for PCR. Different types of positive controls can be run in parallel with the samples: positive controls to test the primers, the reaction and the template can be included depending on the experiment.

6. PCR Amplification of 16S rDNA gene Materials and Method: Prepare master mix for the number of samples plus –ve , +ve controls, plus one extra. - ve control, 1µl of SDW instead of DN +ve control, a DNA ample which is known to give a product of the correct size PCR reaction mix for one 50 µl reaction: (KEEP ON ICE) 10 X NH4 Buffer 5.0 µl dNTP mix (12.5 mM) each 0.8 µl Forward primer 27f (20 µM) 1.0 µl Reverse primer 1525r (20 µM) 1.0 µl MgCl2 3.0 µl Sterile Milli-Q water 38.0 µl Taq polymerase 0.5 µl Prepare a master mix in microfuge tubes and aliquot into PCR tubes . Add 1 µl Genomic DNA (20-200 ng) as template Mix well and microfuge briefly (5 sec pulse) Set the following conditions on PCR thermal cycler 95 oC 5 mins x 1 cycle 95 °C 45 sec 46 °C 30 secs Cycles x 30 cycles 74 °C 45 secs 95 oC 10 mins x 1 cycle