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Blotting techniques.pptx
- 1. Blotting techniques Southern Blotting Subject: Advances in Medical Lab Technology Muhammad Jawad Ullah Lecturer (H.sc) CUSIT
- 2. Blotting Blotting is used in molecular biology for the identification of proteins and nucleic acids and is widely used for diagnostic purposes. It is based on hybridization technique to find out specific sequence or gene of interest or proteins. Nucleic acid molecules such as DNA, RNA and proteins are separated via gel electrophoresis and then transferred to a special nitrocellulose membrane where after hybridization they can be observed. Blotting is performed by allowing a mixture of molecules of interest pass through a block of gel which separates the molecules based on their molecular sizes.
- 3. Southern Blotting Southern blotting was introduced by Edwin Southern in 1975 as a method to detect specific sequences of DNA in DNA samples. The process starts from electrophoresis of DNA molecules which are hybridized in a blotting membrane followed by a transfer step where DNA from gel is transferred onto the blotting membrane. Southern blotting was applied in many important studies like the genetic mapping of the human genome which was based on blotting based detection of restriction fragment length polymorphisms
- 4. Principle Restriction endonucleases, which is an enzyme, is used to break the DNA into small fragments. These fragments are then separated using electrophoresis. The fragments achieved is then classified according to their size (kDa). Thus, DNA fragments are transferred to the blotting paper where it is incubated with probes. Probes used in the Southern blotting can be highly selective.
- 5. Procedure Step 1 DNA Purification To extract the DNA present inside the nucleus of a cell, we must first lyse the entire cell Incubating the cell culture with detergent lyses the entire cell. Now the lysed sample contains DNA, protein, and debris. Protein is lysed by adding the proteinase enzyme and incubated. DNA is purified and separated by alcohol precipitation and fibers are removed by using a buffer. Apart from standard manual isolation procedures, there are commercially available kits for DNA extraction
- 6. Continue… Step 2 Fragmentation The long nucleotide sequences should be broken into smaller fragments for the purification or identification process. This can be done by restriction Endonucleases The components are added to the multiwell plates or microcentrifuge tubes (PCR tubes) and mixed The incubation of the digests is carried out at 37 °C either in an incubator or a water bath.
- 7. Continue… Step 3 Electrophoresis Nucleic acids are negatively charged molecules. So they move towards the anode in an electrophoresis chamber. The DNA fragments having smaller size can move faster than that of large size fragments and enables us to separate them according to the size. Step 4: Denaturation DNA thus attained are double stranded in nature. For our purpose of probe hybridization, we need a single stranded DNA. DNA is therefore denatured in an alkaline solution. This results in the formation of denatured DNA.
- 8. Continue… Step 5 Blotting Blotting is the transfer of the fragmented DNA sequence to the nitrocellulose membrane or nylon membrane. In the original protocol nitrocellulose membrane have been used for the blotting in case of Southern blot but in recent times nylon membranes have been implemented for the blotting process due to their ability to bind more amount of DNA efficiently which allows the Southern blot to be carried out with less amount of target DNA. Gel is soaked in 1.5 M NaCl, 0.5 M NaOH. then nitrocellulose membrane is placed on the gel with weight over it which will result in the transfer of DNA to nitrocellulose membrane by capillary action.
- 9. Continue… Step 6 Hybridization Labeled probe is added to the membrane buffer and incubated for as It takes several hours for the probe to find the exact target sequence. In hybridization labeled probe is complementary DNA radioactively labeled which when shows an complementarity with the single stranded taget DNA on membrane will be detected. The Visualization occurs by exposing it to the X rays that will detect the radiolabeled DNA, if it is Attached to target DNA , a spot will appear on the X ray film and will confirm the result.
- 11. Advantages Effective way to detect a specific DNA sequence in a large, complex sample of DNA. Can be used to quantify the amount of the present DNA. Cheaper than DNA sequencing
- 12. Disadvantages More expansive than most other tests. Complex and labor-intensive. Time consuming and cumbersome.
- 13. Applications To identify specific DNA in a DNA sample. To Isolate desired DNA for construction of rDNA. Identify mutations, deletions, and gene rearrangements. Used in prognosis of cancer and in prenatal diagnosis of genetic diseases. In RFLP. Diagnosis of HIV-1 and infectious disease. In DNA fingerprinting: Paternity and Maternity Testing Criminal Identification and Forensics Personal Identification