2. Blotting
It is the transfer of macromolecules like proteins and nucleic acids from gel onto
surface of immobilized membrane similar to gel.
These powerful techniques allow us to identify and characterize specific
molecules in a complex mixture of related molecules.
3. Types of Blotting
Different blotting is used to detect different type of macromolecules such as
Southern blotting is used for DNA analysis
Northern blotting is for RNA analysis
Western blotting is for protein analysis
Eastern for carbohydrate detection
4. Southern Blotting
1) Southern blotting is a laboratory technique used to detect a specific DNA
sequence in a blood or tissue sample.
2) A restriction enzyme RE is used to cut a sample of DNA into fragments that are
separated using gel electrophoresis.
3) The DNA fragments are transferred out of the gel to the surface of a membrane.
4) The membrane is exposed to a DNA probe labeled with a radioactive or
chemical tag.
5) If the probe binds to the membrane, then the probe sequence is present in the
sample.
5. Steps in Southern Blotting
Step 1 Extract and purify DNA from cells
Step 2 DNA is restricted with restriction enzymes
Step 3 Separate by Gel electrophoresis
Step 4 Denature the DNA by neutralizing its pH
Step 5 Transfer to nitrocellulose paper
Step 6 Blotting.
Step 7 add labelled Probe for hybridization to take place
Step 8 Washing of unbound probe
Step 9 Detection by Autoradiograph
6.
7.
8.
9. Applications
Gene discovery, Mapping, evolution & Development studies, Diagnostics and Forensic
To identify specific DNA in a DNA sample
To isolate desired DNA for construction of rDNA
Identify mutations, Deletions and gene re arrangements
Used in prognosis of cancer and in prenatal diagnosis of genetic disease
Polymorphism
Allow for determination of molecular weights of restriction fragments
Presence of particular bit of DNA in the sample
In RFLP
Diagnosis of HIV-1 and infectious disease
In DNA fingerprinting (Paternity and Maternity testing, Criminal Identification and Forensics, Personal
identification)
10. Northern Blotting
RNA isolation (total or poly(A) RNA)
Probe generation.
Denaturing agarose gel electrophoresis.
Transfer to solid support and immobilization.
Prehybridization and hybridization with probe.
Washing.
Detection.
Stripping and reprobing (optional)
11.
12. Purpose of Northern Blotting
A northern blot is a laboratory method
Used to detect specific RNA molecules among a mixture of RNA.
Northern blotting can be used to analyze a sample of RNA from a particular
tissue or cell type in order to measure the RNA expression of particular genes.
