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BIOL 367 Chapter Notes -Thermal Cycler, Buffer Solution, Methylenetetrahydrofolate Reductase


Department
Biology
Course Code
BIOL 367
Professor
Luc Varin

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2ND YEAR MT MOLECULAR BIOLOGY LAB 2010
Prepared by: Ms. Thoraia Shinawi
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.
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2ND YEAR MT MOLECULAR BIOLOGY LAB 2010
Prepared by: Ms. Thoraia Shinawi
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 9095 °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 5065 °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.
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