blotting technique in biotechnology .pptx

blotting technique in biotechnology .pptx

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  Blotting technique Presentation by SayyedHamid Dept. of pharmacognosy
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  Introduction • What isBlot ? • Meaning: spot or by pressing something soft against it in molecular biology and genetics A blot means method of transferring proteins, DNA or RNA, onto a carrier. Carrier: membrane (nitrocellulose PVDF or nylon membrane) • Blotting: a process of transferring proteins, DNA or RNA, on to a membrane usually after electrophoresis for the clear visualization is called as blotting.
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  Why blotting isdone? • For Visualization of specific DNA , RNA & protein among many thousands of contaminating molecules requires the convergence of number of techniques which are collectively termed BLOT transfer . • Visualized by 1. Colorant staining (for example, silver staining of proteins), 2. Auto-radiography visualization of radioactive labelled molecules (performed before the blot), or 3. specific Labelling of some proteins or nucleic acids.
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  Types of blotting •Southern blot • Western blot • Far-Western blot • Southwestern blot • Eastern blot • Far-Eastern blot • Northern blot • Reverse Northern blot
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  Southern Blotting • In1975 Edward Southern developed this technique that is widely used to detect fragments of DNA . • Definition: Southern blot a method for transferring DNA from an agarose gel to nitrocellulose filter , on which the DNA can be detected by suitable probe ( e.g. : complementary DNA or RNA )
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  • This processinvolves : 1 ) Separation of DNA or DNA fragments by agarose gel electrophoresis . 2 ) DNA fragments are blotted onto a strip of nitrocellulose or a nylon membrane. 3 ) Identification by hybridization with a labelled , complementary nucleic acid probe.
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  • Procedure: 1. TheDNA sample is digested by restriction endonucleases , producing small fragments & that are suitable for analysis . 2. Fragments are separated on agarose gel by electrophoresis. 3. The agarose gel is soaked in a solution of dye (ethidium bromide) & washed for remain excess dye . 4. illumination of the rinsed slab with UV light reveals red orange stains where nucleic acids are located 5. Soak in weak acid to to depurinate & fragment the DNA followed by Denaturation with strong base& neutralisation 6. Transfer on the paper (nitrocellulose) by pressing against gel. During transfer fragmented one strand stick to paper 7. Put the membrane in complementary strand containing solution to make hybrid DNA 8. Wash to remove unbinded
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  Northern blotting Northern blottingis a technique for detection of specific RNA sequences . Developed by James alwine &George stark.  RNA molecules have defined length &much shorter than genomic DNA it is not necessary to cleave RNA before electrophoresis . RNA is more susceptible to degradation than DNA RNA sample are separated based on size by gel electrophoresis .
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  1. RNA isblotted on to a nylon positively charged membrane. 2. The membrane is placed in a hybridization buffer with a labeled probe ( usually DNA ) 3. Labeled probe is detected by autoradiography 4. Expression patterns of sequences of interest in different samples can be compared . Process :
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  Applications A standard fordirect study of the gene expression at the level of mRNA . Detection of mRNA transcript size . Study of RNA splicing – can detect alternatively spliced transcripts . Study RNA half life
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  Disadvantages Time consuming procedure. RNA samples can be degraded by RNases . Use of radioactive probes . Detection with multiple probes is a problem .