By inventing many techniques, biotechnology has made it easy for the scientists to study, DNA or proteins at the molecular level. All the techniques of biotechnology are used for different purposes and perform different functions. A technique which I want to describe is very easy and within less time gives very good results. Polymerase Chain Reaction is one of many techniques and it is used to make different copies of DNA from a single strand. It is defined as creating numerous copies of DNA from a single strand of DNA. It is a technique of molecular biology which is also used in DNA finger printing. This technique is also used in genetics, laboratories of forensic science, paternity testing and other many hereditary diseases.
Polymerase Chain Reaction was first developed by Kary Mullis. It is now used widely for various genetic purposes like finger printing etc. diagnosing the hereditary diseases is also the main concern of PCR. This technique when produces multiple copies of DNA molecules, detect the defected gene which causes disease. Infectious diseases can also be diagnosed with the polymerase chain reaction.
The name of the polymerase chain reaction derived from the DNA polymerase. Through the process of replication many copies of original DNA molecules are made and are studied in various applications. The process is named chain reaction because, after one period of replication, DNA molecules are replicated again, when this replication completes, another period or third cycle of replication starts which makes more replica of DNA molecules. Similarly the process continues until the desired number of copies if DNA molecules are obtained. Polymerase Chain Reaction makes it possible to intensify a single small piece of DNA or the very small parts of DNA can also be replicated. As a result of this whole reaction, millions of copies of DNA molecules are produced. Now a day, polymerase chain reaction is used to perform many functions like, genetic manipulations, diagnostic tests and many other purposes.
The process of polymerase chain reaction depends totally on the thermal cycling. Because during this process, heating and cooling is performed to melt the molecule of DNA repeatedly. Enzymatic replication of the DNA molecule also takes place in the thermal cycling. Primers are the short fragments of DNA sequences which contain the complementary sequences for the target region and also contain the DNA polymerase which has given the name polymerase chain reaction. Primers and DNA polymerase are the two main components of the chain reaction which help in repetitive amplification of DNA molecules. When the new DNA molecules are produced, they can also be used as templates for replication in the reaction. This is how process continues.
Heat stable DNA polymerase is usually used in all the polymerase chain reactions for example Taq polymerase. Thermos aquaticus is a bacterium which is the source of giving this enzyme. A new DNA strand is assembled by this DNA polymerase.
Steps involved in Polymerase Chain Reaction:-
Polymerase chain reaction uses small pieces of DNA molecules and replicates it in the repeated cycles of replication process. Usually a DNA fragment of 10 kilo base pairs is used in the PCR because small pieces of DNA are easy to handle and show good results within less time. Some techniques also amplify 40 kilo base pair fragments of DNA which are little advanced. Basically PCR entails several mechanisms and reagents which are as follows.
1) DNA template is the most important component of the process. Whole mechanism depends on DNA fragments.
2) Two primer are needed which are complementary to each other.
3) Taq polymerase is also needed which have the temperature of 70 degree centigrade.
4) Most important component of the PCR is Deoxynucleoside triphosphates, which helps the DNA molecule to synthesize new strand of DNA.
5) Providing suitable chemical environment to the DNA strand is also very important because then the molecule will not replicate properly. Buffer solution is needed for this purpose.
6) Divalent cations such as manganese are also needed during the PCR because then error rate will be decreased.
Thermal cycle is used for heating and cooling of the DNA molecule melting. Now modifications have been made in the thermal cycle. It will be possible now to heat and cool the reaction by reversing the electric current. Paltier effect is used fro this purpose.
Procedure of running a polymerase chain reaction is as follows:
1) First the reaction is heated at the temperature of 94 to 96C. This temperature is kept for some time. This temperature is only required to heat up the DNA polymerases.
2) In the second step, heat the reaction up to 94 to 98C and this is the first cycle of heating the reaction for 30 minutes. This step is called denaturation step. DNA molecule melts through this heating and disrupts the hydrogen bonds which are present between complementary bases. Thus single strands of DNA are obtained by this heating.
3) The step of annealing involves in lowering the temperature to 65C for a fix period of time. This step allows the primers of DNA to anneal and these strands of DNA convert into single templates. During this step of reaction, hydrogen bonds between single DNA templates are produced when the primer sequences come closer to each other. This is how DNA synthesis commences.
4) The use of DNA polymerases decides the temperature of this step. At the temperature of 75-80C, the enzyme of taq polymerase shows its best activity. In this step, DNA strands are produced which are synthesized from the DNA polymerase in the direction of 5 to 3 n the template.
5) This step is performed just to make sure if all the single strands of DNA are fully extended or not. This process is performed at the temperature of 70C for 15 minutes.
6) The final step is performed to store the reaction, so that it acts well when needed.
Polymerase chain reaction involves in the detection of diseases like leukemia. It uses the DNA samples to detect malignant cells and making corrections in them. PCR is also used for the identification of the micro-organisms which do not cultivate correctly. For example, viruses, bacteria and other animal models by making tissue culture assay.
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