Polymerase Chain Reaction

Before PCR Before PCR Recombinant Recombinant DNA DNA technology technology

PCR Polymerase chain reaction Polymerase chain reaction Definition Definition –Technique for replication or copying a portion of a DNA strand outside a living cell

Some applications of PCR Forensic medicine. Forensic medicine. Pre implantation Genetic Diagnosis (PGD). Pre implantation Genetic Diagnosis (PGD). Archeology. Archeology. Paternity testing. Paternity testing.

A cycle of PCR consists of three steps. DNA denaturation at 94 degrees C. DNA denaturation at 94 degrees C. Primer annealing at 54 degrees C. Primer annealing at 54 degrees C. DNA extension by a thermostable DNA polymerase at 72 degrees C. DNA extension by a thermostable DNA polymerase at 72 degrees C.

Starting with a single molecule of DNA, about 30 rounds or cycles of PCR will produce about 10 million identical DNA molecules!!

PCR, as currently practiced, requires several basic components. These components are: DNA template that contains the region of the DNA fragment to be amplified One or more primers, which are complementary to the DNA regions at the 5' and 3' ends of the DNA region that is to be amplified

Taq polymerase (or another DNA polymerase with a temperature optimum at around 70°C) (dNTPs) from which the DNA polymerase builds the new DNA Buffer, which provides a suitable chemical environment for optimum activity and stability of the DNA polymerase Generally Mg 2+ is used, but Mn 2+ can be utilized Monovalent cation Pottasium ions

The PCR is carried out in small reaction tubes ( ml volumes), containing a reaction volume typically of μl, that are inserted into a thermal cycler( PCR). This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. Most thermal cyclers have heated lids to prevent condensation on the inside of the reaction tube caps. Alternatively, a layer of oil may be placed on the reaction mixture to prevent evaporation

Primers are short oligonucleaotides, i.e., chemically synthesized, single-stranded DNA fragments — usually only 18 to 25 base pairs long — containing nucleotides that are complementary to the nucleotides at both ends of the DNA fragment to be amplified. These complementary bases in primer and DNA template facilitate annealing of the primer to the DNA template to which the DNA polymerase can bind and begin with the synthesis of a new DNA strand that is complementary to the DNA template Primers

Procedure The reaction mixture consists of The reaction mixture consists of 1.0 µl DNA template (100 ng/µl) 1.0 µl DNA template (100 ng/µl) 2.5 µl of primer, 1.25 µl per primer (100 ng/µl) 2.5 µl of primer, 1.25 µl per primer (100 ng/µl) 1.0 µl Pfu-Polymerase 1.0 µl Pfu-Polymerase 1.0 µl nucleotides 1.0 µl nucleotides 5.0 µl buffer solution 5.0 µl buffer solution 89.5 µl water 89.5 µl water A 200 µl reaction tube containing the 100 µl mixture is inserted into the thermocycler. A 200 µl reaction tube containing the 100 µl mixture is inserted into the thermocycler.

Forensic uses of PCR PCR can be used to amplify DNA from a small amount of cells (about 1000 cells). PCR can be used to amplify DNA from a small amount of cells (about 1000 cells). The amplified DNA from cells can be used in DNA fingerprinting analysis. The amplified DNA from cells can be used in DNA fingerprinting analysis.

DNA fingerprinting using PCR in forensic investigations. DNA is isolated from blood at a crime scene and amplified by PCR. DNA is isolated from blood at a crime scene and amplified by PCR. The amplified DNA is digested with restriction enzymes and resolved on an agarose gel. The amplified DNA is digested with restriction enzymes and resolved on an agarose gel. Southern blot analysis is performed to give a DNA fingerprint. Southern blot analysis is performed to give a DNA fingerprint.

After PCR... DNA Fingerprinting DNA Fingerprinting –Extract DNA –PCR –Electrophoresis –Southern Blot  Hybridization: the process of joining 2 complementary strands of DNA to from a double-stranded molecule.

Individuals have unique DNA fingerprints because of restriction length polymorphisms (RFLPs). Many argue that DNA evidence is more reliable than eyewitnesses in placing a suspect at the scene of a crime.