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nucleic acid hybridization

blotting

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nucleic acid hybridization

  1. 1. TITLE-NUCLEIC ACID HYBRIDIZATION -Presented by Miss Pragati Randive
  2. 2. OVER VIEW  Introduction  Southern Hybridization  Northern Hybridization  Western Hybridization  Dot Hybridization  Colony Hybridization
  3. 3. INTRODUCTION  What is DNA hybridization?  Principle and basic procedure  DNA probe  Detector systems
  4. 4.  What is Hybridization? Hybridization is a process of establishing non- covalent, sequence- specific interaction between two or more complementary strands of nucleic acids into a single hybrid.
  5. 5.  Principle : Single stranded DNA molecule recognize and specifically bind to a complementary DNA strand in a mixture of other DNA strand.  Basic Procedure: • Single stranded target DNA is bound to a membrane support. • DNA probe labeled with detector substance is added • DNA probe pairs with the complementary target DNA • Sequence of nucleotide in the target DNA can be identified.
  6. 6.  DNA probe/Gene probe: Synthetic single stranded DNA molecule that can recognize and specifically bind to a target DNA by complementary base pairing in a mixture of bio molecule. Detector system in DNA hybridization: • Radioactive Detector system • Non-Radioactive Detector system
  7. 7.  Non radioactive detector system: This detector system is based on the enzymatic conversion of a chromogenic substrate or chemiluminiscent substrate. Biotin labeled nucleotide are incorporated into the DNA probe.  Procedure for chemiluminiscent detection: • Biotin labeled DNA probe is hybridized to the target DNA. • The egg white protein avidin is added to bind Biotin. • Then biotin labeled enzyme such as alkaline phosphatase is added to attach to avidin, this protein have 4 separate biotin binding site that can bind to biotin labeled enzyme and biotin labeled DNA probe. • On addition of a chemiluminiscent substrate, alkaline phosphate converts it to a light emitting substrate. Procedure for chromogenic detector: Here enzyme peroxidase is added in place of alkaline phosphate, and when a chromogenic substrate is added color is produced which can be measured.
  8. 8. Detecting nucleic acids with non- radioactive probe:
  9. 9. Nucleic acid blotting technique: Blotting refers to process of immobilization of sample nucleic acid in solid support. The blotted nucleic acids are then used as target in the hybridization experiment for their specific detection. Types of blotting techniques: • Southern blotting • Northern blotting • Western blotting • Colony blotting • Dot blotting
  10. 10. • Southern Blotting • First developed by E.M.Southern • DNA-DNA hybridization is the basis. • Later this techinque was extended to RNA(northern blotting) and protein(Western Blotting). Fig: Procedure for southern blotting
  11. 11. Northern Blotting
  12. 12. • Following the separation of the protein mix the polypeptide bands are transferred to a membrane carrier. For this purpose the membrane is attached to the gel and this so-called sandwich is transferred to an electrophoresis chamber. • It is possible that some of the SDS is washed out, and the protein partially re- natures again, i.e. regains its 2D- and 3D structure. However, the applied electric charge causes the proteins to travel out of the gel vertically to the direction they traveled in on the gel, onto the membrane. • The protein bands are thereby bound to the membrane. The "blotted" bands are now available to be treated further (e.g. for detection of specific proteins with specific antibodies). Western Blotting
  13. 13. Immunodetection • The identification of specific antibodies is possible after the separation and blotting of proteins. • Specific antibodies (mono- or polyclonal) bind to "their" band of proteins. Unspecifically binding antibodies are removed by washing with detergent- containing buffers. • Additionally, unspecific binding pockets can be blocked before the addition of specific antibodies. • Primary antibodies are usually applied first, which are then recognized by a secondary antibody. •The secondary antibody is conjugated with colour, radioactivity or an enzyme for detection. Biotin-conjugated antibodies are also used for this purpose Western Blotting
  14. 14. Fig; Illustration of Western Blot immunodetection
  15. 15. Dot Blot The sample DNA or RNA from different individuals are fragmented on to a nitrocellulose filter in the form of dots. The DNA is then denatured and the filter is baked at 80ºc to fix the DNA firmly to the filter. The filter is pre treated to prevent non-specific binding of the probe to the filter. The filter is then treated with appropriate radioactive single stranded DNA probe under condition favouring hybridization. Filter is then washed repeatedly washed to remove free probe. The hybridized probes are detected by autoradiography.
  16. 16.  Colony blotting Fig: Procedure for colony blotting
  17. 17. Southern Blotting Northern Blotting Western Blotting Molecule detected DNA mRNA Protein Gel electrophoresis Agarose Gel Formaldehyde agarose gel Polyacrylamide gel Gel pretreatment Depurination, Denaturation and deneutralization - - Blotting method Capillary transfer Capillary transfer Electric transfer Probes DNA radioactive or nonradioactive cDNA,cRNA, radioactive or nonradioactive Primary antibody Detection system Autoradiography Chemiluminescent colorimetric Autoradiography Chemiluminescent colorimetric Chemiluminescent colorimetric Comparison of Southern, Northern and Western Blotting Techniques

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