Polymerase Chain Reaction (PCR)

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A short and easy collection of knowledge for students. to get power point format copy E-mail (kayani.irfan@gmail.com)

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Polymerase Chain Reaction:

Polymerase Chain Reaction Presented By:- Hafiz Irfan shabbir Kayani CMLT, National Institute of Health Islamabad Pakistan ( kayani.irfan@gmail.com )

Introduction:

Introduction The polymerase chain reaction ( PCR ) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA. First described in 1985, Nobel Prize for Kary Mullis in 1993. The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the bacterium Thermus aquaticus that was discovered in hot springs.

Application of PCR:

Application of PCR DNA cloning for sequencing. DNA based phylogeny, or functional analysis of genes. Diagnosis of hereditary Diseases. Identification of genetic fingerprints. Detection and diagnosis of infectious diseases.

Principal & Procedure:

Principal & Procedure PCR is used to amplify a specific region of a DNA strand. The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction.

Thermal Cycler:

Thermal Cycler

A strip of eight PCR tubes, each containing a 100 μl reaction mixture:-:

A strip of eight PCR tubes, each containing a 100 μl reaction mixture:-

Cycling steps:

Cycling steps There are three major steps in a PCR, which are repeated for 20 to 40 cycles. This is done on an automated Thermal Cycler , which can heat and cool the reaction tubes in a very short time. Denaturation:- At around 94°C (During the denaturation, the double strand melts, open to single stranded DNA)

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Annealing:- At around 54°C ( Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. If the primers exactly fit the template, the hydrogen bonds are so strong that the primer stays attached) Extension:- At around 72°C (The bases (complementary to the template) are coupled to the primer on the 3' side. The polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)

Different steps of PCR:

Different steps of PCR

PowerPoint Presentation:

Every cycle results in a doubling of the number of strands of DNA present . After the first few cycles, most of the product DNA strands made are the same length as the distance between the primers . The result is a dramatic amplification of a the DNA that exists between the primers. The amount of amplification is 2 raised to the n power; n represents the number of cycles that are performed. After 20 cycles, this would give approximately 1 million fold amplification. After 40 cycles the amplification would be 1 x 10 12

(Polymerase chain reaction):

(Polymerase chain reaction)

Loading on gel:

Loading on gel

Quantification/Real-time PCR:

Quantification/Real-time PCR Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample—a technique often applied to quantitatively determine levels of gene expression. Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.

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