09-19-2010, 04:10 PM
(This post was last modified: 09-20-2010, 04:05 AM by Administrator.)
Polymerase chin reaction is a technique of amplifying a single copy of DNA. Thousands and millions of copies of DNA can be produced through this technique having particular sequences. Many primers, enzymes and other conditions are involved in this process. It is often used in DNA fingerprinting, DNA profiling and other tests which are necessary. Its applications range from research to the commercial sector.
Steps Involved in PCR:-
To initiate the polymerase chain reaction it is necessary to have some things like DNA polymerases, Restriction enzymes, a DNA template, primers or short DNA sequences to start the DNA polymerase. Without which the task of making multiple copies cannot be accomplished.
Denaturation:-
The reaction mixture is heated at the temperature of 94 to 98 degree Celsius for 20 to 30 minutes. It makes the double stranded DNA to melt by breaking the hydrogen bonds that keep the two templates together. Now DNA molecule becomes single stranded.
Annealing:-
After the separation of the DNA strands, temperature of the reaction mixture is lowered and is kept at 50 to 65 degree Celsius for 20 to 40 seconds. It causes the annealing of the primers to the single stranded DNA molecules.
Extension:-
In this step, the DNA polymerase makes a complementary strand against each single stranded DNA. Usually Taq polymerase is used for this purpose.
Elongation:-
Usually a temperature of 70 to 74 degree Celsius is required for this step to make sure that all the single stranded DNA templates have found their complementary strands.
Uses of PCR:-
The use of polymerase chain reaction is important in many scientific fields like genetics, genetic engineering and molecular biology. In the disciplines like microbiology and molecular biology, it is used for DNA cloning procedures, DNA sequencing, and recombinant DNA technology in the research laboratories. In clinical microbiology, many microbial infections are diagnosed by PCR. Similarly, epidemiological studies also make use of this technique. Field of forensics is another important field which uses PCR for the identification of criminals and to identify the paternity of the child in the court of justice by obtaining only small amount of blood.
Importance of PCR in Biotechnology:-
Polymerase chain reaction is of vital importance in biotechnology. It can be used for the diagnosis of diseases like AIDS, middle ear infection, Lyme disease and tuberculosis. PCR identifies and cultures those microorganisms which are responsible for causing these diseases. This technique also helped a lot during human genome project in the isolation, amplification and sequencing of human genes. Recombinant DNA technology also makes use of the PCR for making multiple copies of transgenes for different applications for example for diagnosing the diseases and for making certain changes in the genome of an organism.
Disadvantages of PCR:-
Like any other reactions, reaction occurring in the PCR also faces some problems. Usually the polymerases used in PCR do not contain the 3’ to 5’ exonuclease activity. Due to this reason, they are not able to correct any errors while incorporating the nucleotides.
Steps Involved in PCR:-
To initiate the polymerase chain reaction it is necessary to have some things like DNA polymerases, Restriction enzymes, a DNA template, primers or short DNA sequences to start the DNA polymerase. Without which the task of making multiple copies cannot be accomplished.
Denaturation:-
The reaction mixture is heated at the temperature of 94 to 98 degree Celsius for 20 to 30 minutes. It makes the double stranded DNA to melt by breaking the hydrogen bonds that keep the two templates together. Now DNA molecule becomes single stranded.
Annealing:-
After the separation of the DNA strands, temperature of the reaction mixture is lowered and is kept at 50 to 65 degree Celsius for 20 to 40 seconds. It causes the annealing of the primers to the single stranded DNA molecules.
Extension:-
In this step, the DNA polymerase makes a complementary strand against each single stranded DNA. Usually Taq polymerase is used for this purpose.
Elongation:-
Usually a temperature of 70 to 74 degree Celsius is required for this step to make sure that all the single stranded DNA templates have found their complementary strands.
Uses of PCR:-
The use of polymerase chain reaction is important in many scientific fields like genetics, genetic engineering and molecular biology. In the disciplines like microbiology and molecular biology, it is used for DNA cloning procedures, DNA sequencing, and recombinant DNA technology in the research laboratories. In clinical microbiology, many microbial infections are diagnosed by PCR. Similarly, epidemiological studies also make use of this technique. Field of forensics is another important field which uses PCR for the identification of criminals and to identify the paternity of the child in the court of justice by obtaining only small amount of blood.
Importance of PCR in Biotechnology:-
Polymerase chain reaction is of vital importance in biotechnology. It can be used for the diagnosis of diseases like AIDS, middle ear infection, Lyme disease and tuberculosis. PCR identifies and cultures those microorganisms which are responsible for causing these diseases. This technique also helped a lot during human genome project in the isolation, amplification and sequencing of human genes. Recombinant DNA technology also makes use of the PCR for making multiple copies of transgenes for different applications for example for diagnosing the diseases and for making certain changes in the genome of an organism.
Disadvantages of PCR:-
Like any other reactions, reaction occurring in the PCR also faces some problems. Usually the polymerases used in PCR do not contain the 3’ to 5’ exonuclease activity. Due to this reason, they are not able to correct any errors while incorporating the nucleotides.
![[+]](https://www.biotechnologyforums.com/images/netpen-pro/collapse_collapsed.png)