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Blotting
Blotting is a method of analysing DNA and RNA. There are two main blotting techniques.
Northern blotting, or Northern hybridization, is a technique for detection of specific RNA sequences to study gene expression. Northern blotting was developed by James Alwine and George Stark at Stanford University and was named such by analogy to Southern blotting, named for biologist Edwin Southern, used to study DNA. Northern blotting involves the following steps:
- Total cellular RNA or mRNA is size-separated by denaturing agarose gel electrophoresis.
- The separated RNA is transferred onto a nylon membrane.
- The RNA is then detected with isotopic or non-isotopic labeled complementary DNA or RNA probe.
A notable difference in the procedure (as compared with the Southern blot) is the addition of formaldehyde in the agarose gel, which acts as a denaturant.
As in the Southern blot, the hybridization probe may be made from DNA or RNA. Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1975. It allows investigators to determine the molecular weight of a restriction fragment, to measure relative amounts in different samples and to locate a particular sequence of DNA within a complex mixture. DNA (genomic or other source) is digested with a restriction enzyme and separated by gel electrophoresis and transferred from an agarose gel onto a membrane which is then incubated with a probe which is single-stranded DNA. This probe will form base pairs with its complementary DNA sequence and bind to form a double-stranded DNA molecule. The probe is labeled before hybridization either radioactively or enzymatically (e.g. alkaline phosphatase or horseradish peroxidase). Finally, the location of the probe is detected by directly exposing the membrane to X-ray film or chemiluminescent methods.
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