Cloning or amplification of selected DNA sequence in a genome a millifold or more in a short time using the enzyme DNA polymerase is known as Polymerase chain reaction or in short PCR.
It is an extremely powerful recent technique used for gene cloning. This reaction needs no living cells during the cloning process. PCR is used to clone a given DNA sequence in vitro. However, one should know the nucleotide sequence of at least one short DNA segment [erfpr,ed/ {CR machines are now available that do this cloning of gene automatically in a few hour when compared to traditional gene cloning using vectors. PCR thus provides short cut method for gene cloning.
For gene cloning using PCR machines, first a short segment of nucleotide sequence that is complementary to the target region or gene is synthesized in vitor. This short segment is known as primer. The double stranded DNA containing the target sequence is denatured by heating to 95°C For 1 minute.The Heat Treatment separates the two strands of the DNA. The primers synthesized are then added to denatured DNA mixture ,when the mixture is still hot. Then the mixture is called for 2 minutes. When cooled, the primers get annealed to the single stranded DNA sequence of target region. DNA polymerase obtained from the thermophilic bacteria, Thermus aquaticus is then added to the mixture along with activated free nucleotides (dA, dT, dG and dC). The mixture is heated for two minutes. The DNA polymerase adds energized nuclotides one at a time to one end of each primer and synthesizes a long strand of complementary DNA.
The end of the first cycle produces 2 copies of the target DNA. The two copies obtained by this way are again denatured by heat treatment to repeat the cycle. The cycle can be repeated many times just by changing the temperature of the reaction. After 10 cycles 1024 copies would have been produced and at the end of 25th cycle millions of copies of the target gene are produced.
Comparison of PCR with traditional gene cloning :
Earlier to study DNA fragment or gene , large material was required . Entire process may take days,weeks or even months. For cloning DNA as starting material required is in microgram quantities. PCR requires starting DNA material only in very small quantities (nanogram quantities only). PCR requires only few hours to amplify target DNA to at least million fold level. PCR is ultra sensitive, gives pure product,is less costly and can be automated.
Variant of PCR:
(1).Standard PCR - Sequences at both ends of target sequences have to be known. Two primers define ends of target and only that part is amplified.
(2). Single sided PCR - Here DNA is rearranged before amplification so that only one primer is needed. This is also known as anchored PCR. It is developed for studying genes that .
(3). Inverse PCR - DNA at primer sites rather than between two primers is amplifies because primer sites which are bracketing may have important sequence like promoter for triggering target gene into action. If the border sequences of a DNA segment are not known and those of vector are known, then encode proteins for which partial sequences are known . The sequence to be amplified may be cloned in the vector and border sequence of vector may be used as primers in such a way that the polymerisation proceeds in inverse directions i.e., away from the vector sequence flanked by the primers and towards DNA sequence of inserted segment. Similarly if the gene sequences as primers for an inverse PCR to amplify the sequences flanking this gene e.g. the regulatory sequences.
In anchored primer,due to use of one primer,only one stand will be copied first after which be copied first after which a poly G tail will be attached at end of newly synthesized strand. Then this new strand synthesis using an anchor primer with which poly C sequence is linked to complement poly G of the template. In the next cycle both the original primer and anchored primer will be used for gene amplification.
Applications of PCR :
Applications of PCR can be citedin different fields like Diagnosis of diseases, DNA fingerprinting, Molecular mapping, Monitoring genetic engineering and gene therapy experiments, study of polymorphism etc...
(1). Detection of pathogens in food,water and other samples can be done with PCR and specific direction is possible inspite of very low number of very low number of organisms.
(2). Sensitive and specific detections of genes for pathogenicity factors can be done by PCR .
e.g. gene for Staphylococcal Enterotoxin, Listeria hemolysin gene, Campylobacter,E.coli in soil chalamydia in throat, etc is reported.
(3). Instant diagnostic test for tuberculosis was developed by Dr.Sitaram using PCR (Anna University).
(4). Detection of AIDS virus itself (direct) instead of detection of antibody of AIDS (indirect) can be done with PCR.
(5). PCR technique is used for prenatal diagnosis of sickle cell anemia (earlier it was carried by Southern blot technique),diagnosis of Phenylketonuria (PKU), β thalassemia, hemophilia, Tay Sach's disease.
(6). PCR can be used for determination of sex of human and livestock embryos,fertilised in vitro before implantation.
(7). Sex linked disorders can also be detected by this method.
(8). Identification of criminals,disputed parentage are some of the problems which can be tackled effectively by DNA fingerprinting through PCR. From a piece of hair,blood stain,seminal fluid having partially degraded DNA etc. Characterization of individuals is possible through DNA amplification via PCR.
(9). Study of genetic profile of animals,human being which lived few thousands years earlier (source mummys) is possible through use of PCR. Evolutionary and cultural linage can be traced.
(10). Genetic and physical chromosome maps can be prepared both in plants and animals using PCR. PCR has also been used for developing molecular markers closely linked to specific genes of economic importance.
(11). PCR will prove extremely useful to monitor a gene in genetic engineering or gene therapy experiments.
(12). One of the important applications of PCR is study DNA polymorphism. It has been used for study of polymorphism and chromosome mapping in bacteria, maize, soyabean, mouse,humans etc.
(13). This method is known as Random Amplification of Polymorphic DNA (RAPD).RAPD is more simple and efficient RELP.
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