Polymerase Chain Reaction (PCR) Practical Of Genetics Polymerase Chain Reaction (PCR)

Objectives Introduce the students to the preparation of the PCR reaction. Examine the PCR products on agarose gel electrophoresis.

PCR PCR is a technique that used to amplify specific regions of a DNA strand (the DNA target) in vitro. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb).

Before PCR, DNA of interest could only be amplified by over-expression in cells and this with limited yield Kary mullis received a Nobel Prize in chemistry in 1993, for his invention of the polymerase chain reaction (PCR).

Components of PCR reaction The solution must include: The template DNA A thermostable DNA polymerase Two oligonucleotide primers Deoxynucleotide triphosphates (dNTPs) Reaction buffer (Tris, ammonium ions (and/or potassium ions), magnesium ions, bovine serum albumin).

DNA Template Extracted from cells or tissues, which need to be amplify. A primer is a strand of short nucleic acid sequences , that serves as a starting point for DNA synthesis.

It is heat-resistant enzyme that obtain from Thermus aquaticus. Taq DNA Polymerase It is heat-resistant enzyme that obtain from Thermus aquaticus. Thermus aquaticus. Is a species of bacterium that can tolerate high temperatures (thermophilic bacteria).

Deoxynucleotide triphosphate (dNTPs) Mixture of dATP, dTTP, dCTP and dGTP DNA building blocks PCR buffer maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme Mg2+ cofactor of Taq DNA polymerase

PCR procedures : steps Each cycle of PCR includes steps for DNA template : Denaturation : 94°C 15 sec_2 min Primer annealing: 40–60°C 15 sec_60 sec Primer extension: 70–74°C 1–2 minutes

First step: Denaturation Denatures the target DNA by heating it to 94°C for 15 seconds to 2 minutes. The two intertwined strands of DNA separate from one another, producing the necessary single-stranded DNA template for replication by the thermostable DNA polymerase.

Denaturation Heating

Second step: primer annealing The temperature is reduced to approximately 40–60°C. At this temperature, the oligonucleotide primers can form stable associations (anneal) with the denatured target DNA and serve as primers for the DNA polymerase. This step lasts approximately 15–60 seconds.

Primer annealing

Third step: primer extension The synthesis of new DNA begins as the reaction temperature is raised to the optimum for the DNA polymerase. For most thermostable DNA polymerases, this temperature is in the range of 70–74°C. The extension step lasts approximately 1–2 minutes.

Extension

The next cycle begins with a return to 94°C for denaturation. At 30 cycles there are 1,073,741,764 target copies (~1×109).