The Polymerase Chain Reaction (PCR)

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Polymerase Chain Reaction (PCR)

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Presentation transcript:

The Polymerase Chain Reaction (PCR) What is PCR? It is an in vitro method for amplification of certain DNA or RNA fragment. It is a technique used daily in molecular biology. It provides a good alternative for many currently uesd methods.

What we need for PCR? For what we use PCR? DNA or RNA as tamplet. Primrs. dNTPs mix ( dATP , dTTP ,dCTP, dGTP). Buffer ( with Mg 1.5-2.5 mM) Polymerase enzyme. Thermocycler. For what we use PCR? Detection of mutation. Forensic medicine. Direct cloning of PCR product. Genotyping and diagnosis of infectious diseases HIV, HBV. Mutagenesis by PCR.

Principle of the PCR The purpose of PCR is to enzymaticaly synthesizing and amplifying defined DNA sequences ( make a huge number of copies of a gene) A PCR reaction include the target DNA , a thermostable DNA polymerase , 2 oligonucleotide primers, deoxynucleotide triphosphates (dNTPs),reaction buffer and magnesium. The component are mixed the reaction is placed in the thermal cycler. The thermal cycler takes the reaction through a serious of different temperatures for varying a mounts of time. This serious of temperatures and time adjustments is referred to as one cycle of amplification.

The cycling reactions There are 3 major steps in PCR, which are repeated for up to 45 cycles. This is done in automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time. 1.Denaturation at (90-95°C) for 15 second to 2 minutes. During denaturation , the double strand helix open to a single stranded DNA (i.e. the two strands of DNA separate from each other and produce the necessary single stranded DNA template for the thermo stable polymerase.

2.Annealing at (40-72°C) for 30 to 60 seconds. the primer will anneal to the single strand of DNA. The 1st primer is complementary to the beginning of the original strand sequence. The 2nd primer is complementary to the end of the sequence (One from up and the other from down) The two Oligonucleotides primer: is short sequence of DNA nucleotide (20-35 nucleotides) that is complementary to the sequence on one strand of the DNA double helix at the opposite ends of the origin to be amplified.

3. Extension or elongation (at 72°C) for 1 to 2 minutes The bases (complementary to the template ) are coupled to the primer on the 3’side( the polymerase add dNTPs from 5’ to 3’, reading the template from the 3’to5’side , bases are add complementary to the template) This step complete the cycle and the next cycle begins with the return to 95°C for denaturation. The cycle will be repeated so, we will make more amount of DNA from 2 DNA we get 4, 8, 16, and 32….etc.