More Related Content
Similar to @#. Fjc. southern and northen.pdf (20)
More from Keval80 (20)
Recently uploaded (20)
@#. Fjc. southern and northen.pdf
- 1. Southern blotting and Northen blotting techniques
- 2. CONTENTS Blotting Types of blotting Southern blotting Principle Apparatus Steps involved in southern blotting Application Advantages and Disadvantages Northern blotting Steps involved in northern blotting Applications disadvantages
- 3. BLOTTING A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier. The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. Technique for transferring DNA ,RNA and Proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis.
- 5. 1.SOUTHERN BLOTTING A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis -separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named after its inventor, the British biologist Edwin Mellor Southern.
- 6. PRINCIPLE The key to this method is hybridization. Hybridization: It is the process of forming a double- stranded DNA molecule between a single-stranded DNA probe and a single-stranded target DNA. There are 2 important features of hybridization: The reactions are specific-the probes will only bind to targets with a complementary sequence. The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.
- 7. STEPS INVOLVED IN SOUTHERN BLOTTING 1. Extract and purify DNA from cells; 2. DNA is restricted with enzymes;; 3. Separated by electrophoresis; 4. Denature DNA; 5. Transfer to nitrocellulose paper; 6.Add labeled probe for hybridization to take place; 7. Wash off unbound probe; 8. Autoradiograph.
- 8. APPARATUS
- 9. 1.Extract and purify DNA from cells Isolate the DNA in question from the rest of the cellular material in the nucleus. Incubate specimen with detergent to promote cell lysis. Lysis frees cellular proteins and DNA. Proteins are enzymatically degraded by incubation with proteinase. Organic or non-inorganic extraction removes proteins. DNA is purified from solution by alcohol precipitation. Visible DNA fibers are removed and suspended in buffer. 2.DNA is restricted with enzymes.
- 10. 3.Separated by electrophorosis. The complex mixture of fragments is subjected to gel electrophoresis to separate the fragments according to size.
- 11. 4. Denature DNA. The restriction fragments present in the gel are denatured with alkali. This causes the double stranded to become single-stranded. DNA is then neutralized with NaCl to prevent re-hybridization before adding the probe.
- 12. 5.Transfer to nitrocellulose paper. Transfer the DNA from the gel to a solid support, ie, blotting. The blot is made permanent by: Drying at ~80属C Exposing to UV irradiation 6. Add labeled probe for hybridization. The filter is incubated under hybridization conditions with a specific radiolabeled DNA probe.
- 13. The probe hybridizes to the complementary DNA restriction fragment. 7. Wash off unbound probe. Blot is incubated with wash buffers containing NaCl and detergent to wash away excess probe and reduce background.
- 14. 8. Autoradiograph. If the probe is radioactive, the particles emits when expose to X-ray film. There will be dark spots on the film wherever the probe bound.
- 16. 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
- 17. 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. More expansive than most other tests. Complex and labor- intensive. Time consuming and cumbersome. DISADVANTAGES
- 18. 2.Northern Blotting Northern blotting is a technique for detection of specific RNA sequences. Northern blotting was developed by James Alwine and George Stark at Stanford University (1979) and was named such by analogy to Southern blotting.
- 19. Steps involved in Northern blotting 1. RNA is isolated from several biological samples (e.g. various tissues, various developmental stages of same tissue etc.) * RNA is more susceptible to degradation than DNA.
- 20. 2. Samples are loaded on gel and the RNA samples are separated according to their size on an agarose gel . The resulting gel following after the electrophoresis run.
- 21. 3. The gel is then blotted on a nylon membrane or a nitrocellulose filter paper by creating the sandwich arrangement.
- 22. 4. The membrane is placed in a dish containing hybridization buffer with a labeled probe. Thus, it will hybridize to the RNA on the blot that corresponds to the sequence of interest. 5. The membrane is washed to remove unbound probe.
- 23. 6. The labeled probe is detected via autoradiography or via a chemiluminescence reaction (if a chemically labeled probe is used). In both cases this results in the formation of a dark band on an X-ray film. Now the expression patterns of the sequence of interest in the different samples can be compared.
- 24. APPLICATIONS A standard for the study of gene expression at the level of mRNA (messenger RNA transcripts). Detection of mRNA transcript size . Study RNA degradation . Study RNA splicing . Study RNA half-life. Often used to confirm and check transgenic / knockout mice (animals) .
- 25. Disadvantage of Nourthern Blotting 1.The standard northern blot method is relatively less sensitive than nuclease protection assays and RT-PCR. 2. Detection with multiple probes is a problem. 3. If RNA samples are even slightly degraded by RNAses, the quality of the data and quantitation of expression is quite negatively affected.