Polymerase Chain Reaction (PCR) - PROCEDURE & APPLICATIONS - EASY NOTES

PCR invented by Karry Mullis

PRINCIPLE

It is a technique in which a particular sequence of DNA is amplified and a million copies are produced within few hours

REQUIREMENTS:

-- Flanking sequence (region adjacent to the gene of interest either upstream to 5' or downstream to 3') should be known

--Using the flanking sequence, two DNA primers with 20-30 nucleotides which are complementary to the flanking sequence are synthesized.

STEPS OF PCR:

1.Separation/Denaturation

-- The strands of target DNA to be amplified are separated by heating at 95 degrees centigrade for 5 seconds for 2 minutes

2.Priming/Annealing:

-- The separated strands are cooled at 50degrees centigrade for 30 seconds to 2 minutes.

-- The two primers are (one for each strand) are annealed with the complementary single stranded DNA produced in the first step.

3.Polymerization:

--to initiate the synthesis of DNA - dNTP's (nucleotide triphosphates) in excess  , DNA polymerase(Taq Polymerase) are added to the mixture

-- the DNA polymerase used in PCR is heat stable e.g. - Taq Polymerase obtained from bacteria Thermus aquaticus obtained is resistant to high temperatures

--Two new strands complementary to the original single stranded DNA are synthesized.

-- Function of DNA polymerases (Taq polymerase) is to add nucleotides at the 3' end of the primer synthesizing the new strand in 5'-3' direction.

--after one cycle of replication is completed , the mixture is again heated to separate the strands.

-- now there are 4 strands which again bind with the primer and the primer extension is repeated.

-- this polymerization reaction occurs at 72 degrees centigrade for 30 seconds

--Step 1,2,3 are repeated and Repetition is done in an automated instrument called Thermal Cycler .20 -30 cycles are run to produce million copies of DNA

--After amplification, southern blot analysis is done that shows the presence of DNA in the sample.

ADVANTAGES OF PCR:

-- the technique of amplifying DNA is very sensitive and has speed.

--even very less amount of DNA can be amplified and studied.

CLINICAL APPLICATIONS:

1.Diagnosis of Bacterial & Viral Diseases:

- PCR can be used to diagnose even if one bacteria or virus is present in the sample.

e.g corona virus,HIV,

2.DNA Fingerprinting

-- it allows amplification of minute amount of DNA present in blood samples,hair,semen & other body fluids.

--pattern of DNA of an individual is very specific and it is very unique for every person.

3.Diagnosis Of Genetic Disorders:

--it helps in multiplication if genes with mutations.

--e.g sickle cell anemia

4.Prenatal Diagnosis

--helps in amplification of few cells obtained from fetus by amniocentesis & thus helps in diagnosis inherited disorders.

5.Cancer Detection

-helps in monitoring abnormal cells present in cancer treated patients.

-can be used to detect abnormalities of DNA characteristics in cancer