Saturday, 11 April 2020

Southern and Northern Blotting Analysis



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Blotting technique

  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.



Southern blot

  Used to study how genes are organized within genomes by mapping restriction sites in and around segments of genomic DNA for which specific probes are available.
  Combines transfer of electrophoresis -separated DNA fragments to a membrane and subsequent fragment detection by probe hybridization.
    Named after its inventor, the British biologist Edwin Mellor Southern.

Edwin Mellor Southern

  The technique is based on HYBRIDIZATION.
  Hybridization is the process of forming a complementary base-pairing between a single-stranded DNA probe and a single-stranded target DNA.
— The reactions are highly specific i.e. probes will only bind to targets with a complementary sequence.


Principle of hybridization



Transfer methods
  The transfer of electrophoretically separated DNA from gels to two-dimensional solid supports is a key step in Southern hybridization.
  Upward Capillary Transfer: DNA fragments carried from the gel in an upward flow of liquid and deposited on the surface of the solid support
  Downward Capillary Transfer: DNA fragments carried in a downward flow of alkaline buffer and deposited on the surface of a solid support
  Simultaneous Transfer to Two Membranes: For a high concentration of target DNA, the capillary method can transfer DNA simultaneously to two solid supports
  Electrophoretic Transfer: Used for analysis of small fragments of DNA separated by PAGE. Only for nylon membranes
  Vacuum Transfer: DNA and RNA can be transferred rapidly and quantitatively from gels under vacuum

Upward Capillary Transfer
  The liquid is drawn upward through the gel by capillary action.
  Rate of transfer depends on:
      size of the DNA fragments
      concentration of agarose in the gel
  Small fragments of DNA (<1 kb) are transferred within 1 hour
  Larger fragments are transferred more slowly and less efficiently.
  Capillary transfer of DNAs > 15 kb requires at least 18 hours

Set-up for upward capillary transfer


How we do it!
  STEP-I: Isolate genomic DNA (10 μg per lane required)
STEP-II: Restriction digestion with one or more enzymes and agarose gel electrophoresis


  STEP-III: The digested DNA fragments are denatured in the presence of alkali in situ (in gel)
  STEP-IV: The fragments are then neutralized with NaCl to prevent renaturation before the addition of probe
  STEP-V: Upward capillary transfer of DNA fragments to the membrane (solid support). Usually, a nitrocellulose or nylon membrane is used.
  STEP-VI: UV cross-linking of the transferred DNA fragments to the membrane
  STEP-VII: Preparation of radiolabelled probe usually with α-P32 labelled dCTP or dATP. Fluorescence labeling with Digoxigenin (DIG) is also possible.


  STEP-VIII: Hybridization of the membrane with the labeled probe under specific conditions. The probe hybridizes with the complementary DNA fragment.
  Before hybridization blocking is done with salmon sperm DNA to eliminate non-specific probe binding
  STEP-IX: Wash the unbound probe.
  STEP-X: Detection by autoradiography


A typical Southern blot



Application of Southern Blot
·         Primary usage is to identify a specific DNA in a DNA sample.
  To confirm integration of a transgene in the host genome.
  To identify copy number of the transgene integrated in the host genome.
Other applications:
  Identify mutations, deletions, and gene rearrangements.
  In RFLP
  Used in the prognosis of cancer and in prenatal diagnosis of genetic diseases.
  Diagnosis of HIV-1 and infectious disease.
   In DNA fingerprinting:
      Paternity and Maternity Testing
      Criminal Identification and Forensics
      Personal Identification









Northern Blot Analysis

     Northern blotting is a technique for the detection of specific RNA sequences in the transcriptome.
      Northern blotting was developed by James Alwine and George Stark (1979) and was named such by analogy to Southern blotting.



Application of Northern Blot
  Used 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 transgene expression.




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