Brief Notes on Polymerase Chain Reaction (PCR)

Transcription

1 Brief Notes on Polymerase Chain Reaction (PCR) What is Polymerase Chain Reaction? It is a fast and inexpensive technique used to amplify small and targeted segments of DNA to produce million of copies, sometimes called "molecular photocopying" of a specific gene fragment. What is PCR technique used for? It is a technique used to study the molecular pathogenesis and diagnosis of a variety of acquired, inherited, viral and bacterial diseases. PCR Components: A basic PCR technique requires certain components and reagents that include: 1. Two primers (Forward & Reverse): short pieces of artificially prepared DNA that will target the gene fragment of interest in the entire genome. They are complementary to the 3 ends of each strand of the double stranded target gene i.e. DNA. 2. Taq polymerase (DNA polymerase): It s an enzyme whose function is to extend the new DNA strand. Taq polymerase attaches near the end of the primer and start adding nucleotides. It requires double stranded DNA to become functional. The DNA polymerase in our bodies breaks down at temperatures below 95 C -- the temperature necessary to separate two complementary strands of DNA in a test tube. The DNA polymerase (Taq polymerase) that's used in PCR comes from a strain of bacteria called Thermus aquaticus that live in the hot springs. It can survive near boiling temperatures and works well at 72 C. 3. Deoxynucleotide triphosphate (dntp s): They are the building blocks from which the DNA polymerases synthesizes a new DNA strand. Taq polymerase grabs nucleotides that are floating in the liquid around it and attaches them to the end of a primer.

2 4. Buffer solution: providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. 5. MgCl 2 : acts as a cofactor for the polymerase enzyme. 6. Extracted DNA sample: containing the target region to be amplified. PCR Steps: PCR is a three-step process which is repeated in several cycles. The three steps are: 1. Denaturation step: This step consists of heating the reaction to C. It causes DNA separation by disrupting the hydrogen bonds between complementary bases, yielding single strands of DNA. 2. Annealing step: The reaction temperature is lowered to C allowing hybridization of the primers to the single-stranded DNA template. 3. Extension/Elongation step: At this step, the Taq polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dntps that are complementary to the template in 5' to 3' direction. This process is repeated as many as 30 or 40 times, leading to more than one billion exact copies of the original DNA segment. The entire cycling process of PCR is automated and can be completed in just a few hours using a machine called a thermal cycler.

3

4 Polymerase Chain Reaction for Methylenetetrahydrofolate reductase (MTHFR) gene. Objective: PCR will be carried out to make millions of copies of a specific DNA fragment consisting of a known functional mutation in the MTHFR gene by using site specif primers. You'll be given the following: PCR Buffer. Deoxynucleotide Triphosphate (dntp). Forward Primer. Reverse Primer. Taq polymerase. Deionized distilled water (dd.h 2 O). Samples of DNA (Negative control, Sample 1, Sample 2, Sample 3). Method: 1. Prepare a single tube called "Master Mix". This "Master Mix" will contain all of the above except the DNA sample. Prepare the "Master Mix" for five reactions according to the following table: (The 5 th reaction is extra for pipetting errors)

5 Volume in µl for one Rx Volume in µl for Five Rxs PCR Buffer 5 5x5=25 dntps 1 1x5=5 Forward Primer 1 1 x5=5 Reverse Primer 1 1 x5=5 Taq Polymerase x5=1 dd.h 2 O x5=199 Total Volume Divide the master mix into four sterile tubes and discard extra mix. Total Volume in Maste Mix = 240 µl Neg. Control Sample 1 Sample 2 Sample 3 3. Add to each labeled tube 2 µl of the corresponding DNA sample so that the final volume in each tube is 50 µl. 4. Close the caps tightly and place the tubes in thermal cycler. Start the thermal cycler on required program. At the end of 33 cycles, you'll be able to get more than one billion of copies of the specific gene fragment containing the mutation or region to be analyzed. Once the cycling is completed, place your tubes at 4 o C for storage.