Thursday, November 27, 2008
Advance Biotech
Polymerase Chain Reaction (PCR)
This technique was developed by Karymullis at Cetus corporation between 1983 & 1985.
PCR provide a simple and ingenous method for amplification of specific DNA sequence by in vitro DNA synthesis . By this technique has made it possible to synthesis the large quantities of a DNA fragment without cloning it.
The details of PCR techniques and its mechanism are described by Erlich (1989) in his edited book PCR Technology .The PCR includes the following three essential steps to amplify a specific sequence.
(i) Melting of target DNA-The target containing sequence between 100 and 5,000 base to be amplified is denatured (around 94 degree centigrade for 15 sec ) to separate its complementary strands .This process is called melting of target DNA.
(ii) Annealing of Primers-The second step is the annealing of two oligonuclotide primers to the denaturated DNA strands. Primers are added in excess and the temperature lowered to about 68 degree centigrade for 60 sec. Consequently the primers from hydrogen bonds i.e. anneal to the DNA on both sieds of the DNA sequence.
(iii) Primer Extention-Finally, nucleoside triphosphate (dATP,dGTP,dctp,dTTP) and a thermo stable DNA polymerase are added to the reaction mixture.The DNA polymerase are added to the reaction mixture.The DNA polymerase accelerated the polymerization process of primers and therefore,extends the primers (at 68 degree centigrade) resulting. In synthesis of copies of target DNA seq. only those DNA polymerase which are therstable i.e. ucton at the high temperature are employed inPCR technique.
For this two popular enzymes are used Taq polymerase ( a thermophilic bacterium thermos aquticus) and vent polymerase-Thermococcus litoralis are used in PCR technology.
Theses enzymes exhibit relative stability at DNA melting replacement after each cycle of synthesis.
Also it reduces the cost of PCR and allow automated thermal cycling.
After completion of step 3 (of one cycle) the targated sequences on both strands are copied and four and four strands are produced. Now the three step cycle is repeated which yields 8 copies from four strands.
Similarly, the third cycle produces 16 strands. This cycle is repeated about 50 times.
20 cycles (each of three steps) will produce about one million copies of the target sequence 30 cycle will produces one billion copies.
For working of PCR about 10-100 picomoles of moles are required.
The con of target DNA can be about 10-20 to 10-20 M or 1-105 DNA copies per Ml.)
PCR machine can carry out 25 cycle and amplify DNA 10 5 times in 75 min. The rTth DNA polymerase will transcribe RNA to DNA. Thereafter amplify the DNA.
Therefore, cellular RNA and RNA virsues may be studied when they are present in small quantities.
Application of the PCR Technology-
1.Amplification of DNA and RNA.
2.Determination of orientation and location of restruction fragments relative to one another.
3.Diagnosis of disease and causal micro-organisms.
For example- PCR based diagnosis test for AIDS,Chlamydia, tuberculosis,hepatitis.
4.The PCR is important in detection of genetic disease such as sickle cell anaemia Phenyketonuria and muscular dystrophy.
5.It is also applied in diagnosis of plant disease. Alarge no. of plant pathogens in various hosts or environmental sample are detected by using PCR, for example viriod.
This technique was developed by Karymullis at Cetus corporation between 1983 & 1985.
PCR provide a simple and ingenous method for amplification of specific DNA sequence by in vitro DNA synthesis . By this technique has made it possible to synthesis the large quantities of a DNA fragment without cloning it.
The details of PCR techniques and its mechanism are described by Erlich (1989) in his edited book PCR Technology .The PCR includes the following three essential steps to amplify a specific sequence.
(i) Melting of target DNA-The target containing sequence between 100 and 5,000 base to be amplified is denatured (around 94 degree centigrade for 15 sec ) to separate its complementary strands .This process is called melting of target DNA.
(ii) Annealing of Primers-The second step is the annealing of two oligonuclotide primers to the denaturated DNA strands. Primers are added in excess and the temperature lowered to about 68 degree centigrade for 60 sec. Consequently the primers from hydrogen bonds i.e. anneal to the DNA on both sieds of the DNA sequence.
(iii) Primer Extention-Finally, nucleoside triphosphate (dATP,dGTP,dctp,dTTP) and a thermo stable DNA polymerase are added to the reaction mixture.The DNA polymerase are added to the reaction mixture.The DNA polymerase accelerated the polymerization process of primers and therefore,extends the primers (at 68 degree centigrade) resulting. In synthesis of copies of target DNA seq. only those DNA polymerase which are therstable i.e. ucton at the high temperature are employed inPCR technique.
For this two popular enzymes are used Taq polymerase ( a thermophilic bacterium thermos aquticus) and vent polymerase-Thermococcus litoralis are used in PCR technology.
Theses enzymes exhibit relative stability at DNA melting replacement after each cycle of synthesis.
Also it reduces the cost of PCR and allow automated thermal cycling.
After completion of step 3 (of one cycle) the targated sequences on both strands are copied and four and four strands are produced. Now the three step cycle is repeated which yields 8 copies from four strands.
Similarly, the third cycle produces 16 strands. This cycle is repeated about 50 times.
20 cycles (each of three steps) will produce about one million copies of the target sequence 30 cycle will produces one billion copies.
For working of PCR about 10-100 picomoles of moles are required.
The con of target DNA can be about 10-20 to 10-20 M or 1-105 DNA copies per Ml.)
PCR machine can carry out 25 cycle and amplify DNA 10 5 times in 75 min. The rTth DNA polymerase will transcribe RNA to DNA. Thereafter amplify the DNA.
Therefore, cellular RNA and RNA virsues may be studied when they are present in small quantities.
Application of the PCR Technology-
1.Amplification of DNA and RNA.
2.Determination of orientation and location of restruction fragments relative to one another.
3.Diagnosis of disease and causal micro-organisms.
For example- PCR based diagnosis test for AIDS,Chlamydia, tuberculosis,hepatitis.
4.The PCR is important in detection of genetic disease such as sickle cell anaemia Phenyketonuria and muscular dystrophy.
5.It is also applied in diagnosis of plant disease. Alarge no. of plant pathogens in various hosts or environmental sample are detected by using PCR, for example viriod.