2ND YEAR MT MOLECULAR BIOLOGY LAB 2010

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.

Prepared by: Ms. Thoraia Shinawi

2ND YEAR MT MOLECULAR BIOLOGY LAB 2010

4. Buffer solution: providing a suitable chemical environment for optimum activity and
stability of the DNA polymerase.
5. MgCl2: 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 90–95 °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 50–65 °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.

Prepared by: Ms. Thoraia Shinawi

2ND YEAR MT MOLECULAR BIOLOGY LAB 2010

Prepared by: Ms. Thoraia Shinawi

2ND YEAR MT MOLECULAR BIOLOGY LAB 2010

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.H2O).

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 5th reaction is extra for pipetting errors)

Prepared by: Ms. Thoraia Shinawi

2ND YEAR MT MOLECULAR BIOLOGY LAB 2010

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 0.2 0.2 x5=1

dd.H2O 39.8 39.8 x5=199

Total Volume 48 240

2. 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 4oC for storage.

Prepared by: Ms. Thoraia Shinawi