environmental microbiology-6

LAB #6 POLYMERASE CHAIN REACTION (PCR)

Introduction Polymerase chain reaction (PCR) amplification of DNA has become a key protocol in many microbiological laboratories. PCR is an enzymatic reaction used to amplify specific DNA sequences. A typical amplification cycle involves denaturing the double-stranded DNA into single strands and extending the primer sequences using a DNA polymerase to complete the synthesis of complementary strands. PCR allows amplification of DNA in vitro. The principle of the methodology involves the repetitive enzymatic synthesis of DNA, using two oligonucleotide primers that hybridize to opposite strands of DNA which flank the target DNA of interest. The primers are often unique 20 – 30 base long oligonucleotides carefully chosen to flank and allow amplification of the target DNA of interest. During each cycle, the number of copies of template DNA is theoretically doubled. In practice, 25 cycles of amplification results in approximately a million-fold increase in the number of DNA copies. There are three steps in PCR amplification:

 Template Denaturation: Denaturation involves the separation of two strands by heating (94 – 100oC), thereby breaking the hydrogen bonds which bind the two strands of DNA together.

 Primer Annealing: The temperature is lowered to 50 – 70oC. The primers hybridize by forming hydrogen bonds. The higher the temperature of annealing is, the more specific the annealing is, and the extent of annealing of mismatched primer to template is reduced.

 Primer Extension: The Taq polymerase can now recognize the primed strands and attach to the double-stranded portion. The optimum temperature for Taq polymerase to work is 72oC. At this temperature, the Taq polymerase will start constructing the complementary strands using the deoxynucleotides (dATP, dCTP, dGTP, and dTTP), which are also present in the solution.

These three steps occur at different, but defined temperatures and time intervals. These cycles are performed in an automated, self-contained temperature cycler, or thermocycler. The temperature cycle allows for precise temperature control required for the initiation of each step. Important points to remember about PCR include:

 It is a cyclical process that produces large amounts of nucleic acids from small quantities of starting material to amplify up to 106 fold or more.

 It is a simple enzymatic reaction that uses DNA polymerase enzyme to copy a target DNA sequence repeatedly during a series of 25 – 40 cycles.

 The essential component of PCR extension is a polymerase enzyme, such as Taq polymerase, which sequentially adds bases to the primer. This enzyme is obtained from thermophilic bacterium called Thermus aquaticus and is uniquely suited for PCR because it is heat stable and can withstand temperatures up to 98oC. This can be reused for many cycles.

 PCR product typically is visualized using agarose gel electrophoresis, and the size of the product is estimated by comparison with DNA standards of known size.

Procedure First Period Materials:

Water sample PCR reaction tubes Micropipettes and the associated tips Master mix materials Sterile water Thermocycler Disposable rubber gloves

Methods:

1. Make a master mix for two PCR reactions in a microfuge tube. The master mix contains the constituents that are necessary for the reaction. These include: deoxynucleotides (dNTPs), 2 primers (forward and reverse), PCR buffer, Taq DNA polymerase enzyme, and DNA/RNA free water. See attached table for reagent volumes.

2. Aliquot 25 l of the master mix into each of the two PCR reaction tubes. Close the tubes and label them “sample” and “(-)”.

3. Add 6 l of sample to one tube. For the negative control, add 6 l of sterile water to the other tube. Use a new pipette tip for each tube.

4. Place the tubes in the thermocycler. The template DNA is initially denatured at 95oC for 5 minutes, followed by 35 cycles of PCR at 95oC for 30 seconds, 45 second of annealing at 60oC, and 30 seconds of extension at 72oC. For the last cycle, the extension step requires 5 minutes to complete synthesis of all strands. The entire process should take approximately two hours.

Second Period Materials:

PCR product from the first period Agarose Laboratory balance (±0.001 g) 40 ml Erlenmeyer flask 40 ml 1X TBE buffer Microwave oven Electrophoresis gel casting tray with comb Microfuge tubes Micropipettes and the associated tips Ficoll loading buffer DNA ladder stock Electrophoresis gel apparatus with electrodes

1 g/ml ethidium bromide Disposable rubber gloves Ethidium bromide liquid and solid disposal containers UV transilluminator UV protective goggles Gel documentation system

Methods: 1. Prepare a 1.6% agarose gel by weighing out 2 grams of agarose in a 124 ml Erlenmeyer

flask and adding 125 ml of 1X TBE buffer. Place in the microwave for 1 minute at high power. Remove from the microwave and swirl to ensure that all of the agarose has melted. If not, reheat for an additional minute.

2. Cool melted agarose to approximately 40oC. Add 6 l ethidium bromide to the gel, and swirl to mix. Ethidium bromide is a carcinogen, so take precautions when handling it, and dispose of all waste in a separate ethidium bromide waste container.

3. Pour molten gel into the electrophoresis gel-casting tray and make sure that no bubbles are trapped within the agarose. Place the comb in the gel and wait for approximately 15- 20 minutes for the gel to solidify. The comb creates wells in the gel to accommodate sample loading.

4. Carefully remove the comb and place the gel in the electrophoresis chamber. Add 1X TBE buffer until the gel is completely submerged.

5. Mix 5 l of loading buffer with 15 l of PCR sample on a piece of parafilm (avoid

pipetting the top layer, which is mineral oil). Carefully load 20 l of each sample into the gel wells.

6. Load 1.5 l of a 100 base pair (bp) DNA ladder, which already contains loading buffer into a separate well.

7. Connect the electrodes to the power source and run the gel under constant current at 90 volts for 1.5 – 2 hours.

8. Turn off the power source, disconnect the electrodes. 9. Place the gel on the transilluminator and observe DNA bands in the presence of UV light.

Photograph the gel. Compare the sizes of the amplified product to the DNA size marker. Questions

1. Explain the temperature profile used for the PCR amplification. What happens at 94, 55, and 72oC? 3 pts

2. What was the size of your amplification product based on a comparison with the DNA ladder? 2 pts

3. What are four ways that you reduced sample contamination throughout the experiment? 2 pts

4. Explain what the following reagents do in a PCR reaction: Taq DNA polymerase, primers, and dNTPs. 3 pts

5. Explain the role of the ethidium bromide in this experiment. 2 pts

POLYMERASE CHAIN REACTION (PCR)

Performing PCR on extracted DNA from E. coli (Amplifying specific region (sequences) of E.

coli DNA)

Reagents Amount

Water 7 µl

Green Master Mix 25 µl

Primers 12 µl

Target (E.coli extracted DNA) 6 µl

Total Volume 50 µl

Place tubes in thermocycler and run the following program:

95o for 5 min

95o for 0:30

60o for 0:45

72o for 0:30

72o for 5 min

Contains

Buffer

dNTPs

Taq polymerase

MgCl2

35 cycles