advance biotech

Southern Blotting-
The name of this technique is derived from the name of its inventor E.M. southern .When DNA-DNA hybridization done it is called Southern Blotting, In this technique DNA is transferred from gel to the nitrocellulose filter.
Procedure-1.DNA sample is digested with restruction enzyme.
2.Digested sample is electrophorased on agrose gel.
3.DNA bands in gel are denaturated by alkali to get single standard DNA.
4.Gel is than nutrilized by socking in tris buffer.
5.The Gel is positioned on top of buffer socked filter and a piece of transfer medium Nitrocellulose or nilone membrane) is placed on top of gel completing the gel sandwhich.
6.Dry filter paper are peeled on top of the filter and a wt put on it.
7.Capalary action pulls the buffer from the bottom filter threw the gel to the transfer medium and up threw the paper a rapid transfer by Appling vacuum also been development.
8. The arragment made for blotting ,is left undistributed for hour or over night and than top filter and wt is removed.
9.Single strand DNA fragment are traped on the transfer medium
DNA fragment fixed permanently to by baking at 80 degree centigrade nitrocellose membrane by baking with U.V. light.
10.DNA fragments on membrane can then be probed for sequence of interest by hybridization membrane is washed to remove unbound DNA.
11. X-ray film is than exposed to the membrane to get autoradiograph.
Application-
The southern blotting technique is extrely sensitive.
i.It can be used to map the restriction sites around a single copy gene sequence in any genome (even of man).
ii.It is used for DNA fingerprinting.
iii.Preparation of RFLP maps.
iv.Detection and identification of the transferred genes in transgenic individuals etc.

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.