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    Southern blotting is a technique used to detect specific DNA sequences. It involves digesting DNA with enzymes, separating fragments by size through gel electrophoresis, transferring DNA from the gel to a membrane, then using a labeled probe to detect complementary DNA sequences via hybridization and visualization. Southern blotting allows identification of target DNA, and has applications in gene mapping, genetic testing, and disease diagnosis, though it is time-consuming.

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    SOUTHERN BLOTTING

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    Southern blotting is a technique used to detect specific DNA sequences. It involves digesting DNA with enzymes, separating fragments by size through gel electrophoresis, transferring DNA from the gel to a membrane, then using a labeled probe to detect complementary DNA sequences via hybridization and visualization. Southern blotting allows identification of target DNA, and has applications in gene mapping, genetic testing, and disease diagnosis, though it is time-consuming.

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    27 views4 pages

    Southern Blotting

    Uploaded by

    umarlucky1819
    Southern blotting is a technique used to detect specific DNA sequences. It involves digesting DNA with enzymes, separating fragments by size through gel electrophoresis, transferring DNA from the gel to a membrane, then using a labeled probe to detect complementary DNA sequences via hybridization and visualization. Southern blotting allows identification of target DNA, and has applications in gene mapping, genetic testing, and disease diagnosis, though it is time-consuming.

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    of 4

    Department: Microbiology And

    Molecular Genetics
    Semester: 8th evening
    Subject: Adv. in microscopic
    technology
    Assignment topic: Southern
    blotting
    Submitted to: Mam Zilursh
    Naeem
    Submitted by: Group no. 06
    Group members:
    Hifza Sultan-5013
    Sunaina Javed-5027
    Aliya Rafique-5031
    Rubab Ashraf-5005
    Mariyam Abbas-5024
    Sheeza Noor-5029
    Submission date:17july,2023.

    SOUTHERN BLOTTING
    DEFINITION
    Southern blotting is a molecular biology technique used for DNA detection,
    characterization, and quantification.
    An example of RFLP(restriction fragment length polymorphism), southern blotting can be
    defined as an analytical technique for identifying specific sequences of DNA by
    separating fragments on a gel and transferring them to a second medium (carrier
    membrane) on which hybridization testing may be carried out.

    PRINCIPLE

    Southern blotting is a molecular biology technique used to detect specific DNA sequences in
    a sample. It is named after its inventor, Edwin Southern, who developed the method in 1975.
    The principle of Southern blotting involves several steps and uses DNA hybridization to
    identify target sequences.

    The main steps in the Southern blotting process are as follows:

    1. DNA Digestion: The first step is to isolate the DNA from the sample of interest, such
    as genomic DNA. The DNA is then cut into smaller fragments using restriction enzymes,
    which recognize specific DNA sequences and cleave the DNA at those sites.

    2. Gel Electrophoresis: The DNA fragments obtained from the digestion are separated
    based on their size using gel electrophoresis. The DNA fragments are loaded into wells of an
    agarose gel and subjected to an electric field. Smaller fragments migrate faster through the
    gel than larger ones, leading to the separation of DNA fragments by size.

    3. DNA Denaturation: After electrophoresis, the DNA fragments are transferred from
    the gel to a solid support, usually a nitrocellulose or nylon membrane. This transfer process is
    called "blotting." Before blotting, the DNA in the gel needs to be denatured, breaking the
    double-stranded DNA into single strands.

    4. Blotting: The transfer of DNA from the gel to the membrane is typically achieved
    through a capillary or vacuum transfer. As a result, the DNA on the membrane retains the
    same pattern of separation as in the gel.

    5. DNA Hybridization: The membrane is then subjected to a hybridization step, where it


    is incubated with a labeled DNA probe. The probe is a single-stranded DNA molecule
    complementary to the target sequence of interest. It is labeled with a radioactive or
    fluorescent tag, allowing its detection.

    6. Washing: After hybridization, the membrane is washed to remove any unbound or


    nonspecifically bound probes.

    7. Detection: The labeled DNA probes that have successfully hybridized with the
    complementary DNA sequences on the membrane can be visualized using autoradiography
    (for radioactive probes) or fluorescence imaging (for fluorescent probes).

    By following these steps, Southern blotting allows researchers to detect the presence or
    absence of specific DNA sequences in a sample and determine their approximate size based
    on their position in the gel. This technique has been widely used in molecular biology for
    various applications, including gene mapping, genetic testing, and gene expression analysis.
    However, it is essential to note that Southern blotting is a time-consuming and labor-intensive
    technique, and it has largely been replaced by more advanced methods such as polymerase
    chain reaction (PCR) and various DNA sequencing technologies for most applications.
    APPLICATIONS

     Identify specific DNA from a DNA sample.


     Identification of viral and bacterial infections.
     Important in the study of gene mutation, deletion and rearrangements.
     DNA fingerprinting (maternity and paternity analysis, forensic studies and personal
    identification).
     Diagnosis of neonatal and genetic diseases including cancer.
     Discovery of RFLP (restriction fragment length polymorphism) to map crucial
    genomes.
    ADVANTAGES AND DISADVANTAGES

    Advantages
     Less degraded compared with protein and mRNA as DNA are very stable.
     Effective way to detect specific DNA sequence from large complex samples.
     Increased sensitivity of fragments detection because of probe label used for amplifying
    signals.
     Only way to diagnose FSHD (Facioscapulohumerals Muscular Dystrophy).
    Disadvantages
     Not applicable in routine diagnostic setting.
     Time consuming and requires large amount of DNA.
     Does not allow morphologic preservation of tissue so historic evaluation features are
    not available.
    REFERENCES

    1. Terry Brown (1993), Analysis of DNA Sequences by Blotting andHybridization


    2. Terence A Brown (2001), Southern Blotting and Related DNA Detection
    Techniques
    3. DaidreeTofano, Ilse R. Wiechers, Robert Cook-Deegan (2006), Edwin Southern,
    DNA blotting, and microarray technology: A case study of the shifting role of
    patents in academic molecular biology
    4. Ed Southern (2006), Southern blotting protocols
    5. Ian A. Hood (2006),The mycology of the Basidiomycetes
    6. Daniela Furrer, Francois Sanschargin, Simon Jacob, Caroline Diorio (2015),
    Advantages and Disadvantages of Technologies for HER2 Testing in Breast Cancer
    Specimens
    7. Manu Tomar(2016), Types of Blotting, Research & Reviews: Journal of
    Pharmaceutics and Nanotechnology

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