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    Southern Blotting

    Uploaded by

    harshita sharma
    Southern Blotting

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    © All Rights Reserved
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    Southern Blotting

    Uploaded by

    harshita sharma
    7 views4 pages
    Southern Blotting

    Copyright

    © © All Rights Reserved

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    DOCX, PDF, TXT or read online from Scribd

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    Southern Blotting

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    7 views4 pages

    Southern Blotting

    Uploaded by

    harshita sharma
    Southern Blotting

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
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    #Blotting: A blotting is a method of transferring DNA, RNA or proteins onto a membrane e.g.

    a
    nitrocellulose, or nylon membrane).

    #Southern Hybridisation:

    • By Edwin Southern in 1975.

    •Southern hybridisation is a technique used for detection of a specific DNA sequence in DNA
    samples.

    •The DNA detected can be a single gene or it can be part of a larger piece of DNA sequence as
    a viral genome.

    •The technique is known as 'DNA transter' or 'southern Blotting'

    •This method involves separation, transfer and hybridization.

    •Southern blotting combines agarose gel electro- phoresis for Size separation of DNA with
    methods to transfer the size separated DNA to a filter membrane for probe hybridization.

    # Principle:

    • The key to this method is hybridization

    • Hybridization-process of forming a double-stranded DNA molecule between a single-


    stranded DNA probe and a single-stranded target DNA.

    # Process:

    Step: 1 DNA isolation & purification:

    For Isolation and purification of DNA from cells


    -Incubate cells with detergent to promote cell lysis.

    -Lysis frees cellular proteins and DNA.

    • Proteins are enzymatically degraded by incubation with proteinase.

    •DNA is purified from solution by alcohol precipitation.

    Step:2 Restriction Digestion:

    -Cut the DNA into different sized fragments using restriction endonucleases (RE) like Eco R1

    Step:3 Gel Electrophoresis

    -Technique for separation of DNA fragments by size

    -Gels are solid Agarose with microscopic pores

    -Gel is soaked in a buffer

    -Standards should also be run

    -Stained with Ethidium bromide for visualization

    Step:4 & 5 Denaturation & Blotting

    -DNA is then denatured with an alkaline solution such as NAOH.

    -DNA is then neutralized with NaCl to prevent re-hybridization before adding the probe.

    -Blotting is the transfer of DNA bands from the gel to a nitrocellulose membrane.

    -Southern blotting set up include: weight <0.75kg; Glass plate; paper tissue; 3 sheets filter paper;
    Membrane; Gel; plastic tray
    -Trnsfer Buffer moves upward by capillary action, so DNA present in gel also moves and
    reaches the membrane and get strongly associated. Fix DNA with heat treatment
    • Hybridization: The labeled probe is added to the membrane in buffer and incubated for several
    hours to allow the probe molecules to find their targets.
    Step:6
    • Probe: Probes are small piece of labeled DNA (attached with detector molecule) used to
    find complementary DNA fragment

    Step 7 & 8: Wash & Autoradiography

    •Washing: Unbound probes are washed out

    • For Radioactive probes, X - ray film is placed over the membrane.


    #Application

    -To identify specific DNA in a sample

    -To Isolate desired DNA for making of rDNA

    -Helps in mapping genes by determining the presence or absence of specific DNA fragments on
    chromosomes.

    -Used to confirm the presence of foreign DNA in genetically modified organisms.

    -Used in prognosis of cancer and in prenatal diagnosis of genetic diseases

    -In RFLP

    - Used in phylogenetic analysis

    -Diagnosis of HIV-1 and infectious disease

    -Applications of DNA fingerprinting include:

    • Paternity and Maternity Testing

    • Criminal Identification and Forensics

    • Personal Identification

    # Limitations:

    -May lack sensitivity when detecting low-copy-number targets, making it less suitable for
    samples with limited DNA.

    -Can be time-consuming compared to more rapid methods like PCR.

    -Often utilized radioactive probes, posing safety concerns. Non-radioactive alternatives, such as
    chemiluminescent or fluorescent probes, have been developed

    -Cross-hybridization can occur, leading to false-positive results if the probe is not highly specific.

    -Depends on the quality and integrity of the DNA sample. Degraded DNA may yield unreliable
    results.

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