Contents 1 Blotting Methods 2 Sir Edwin Mellor Southern 3 Introduction 3.1 Some important definitions for this topic 4 Types of Blotting Methods 4.1 Southern blotting 4.1.1 Procedure 4.2 Northern blotting 4.2.1 Procedure 4.3 Western blotting 4.3.1 Procedure 4.4 Links 4.4.1 Related Articles 4.4.2 External Links 4.4.3 References

edit Blotting Methods

edit Sir Edwin Mellor Southern

Born: 7 June 1938

Carrer: Professor of Biochemistry (Emeritus) at the University of Oxford and a fellow of Trinity College

1998: Royal Medal of the Royal Society of London

2003: in June, was made a Knight Bachelor Birthday Honours for services to the development of DNA microarray technologies

2005: Lasker Award-winning (invention of the Southern blot methods which he started developing in 1975).^[1]

This citation was prepared by Alec Jeffreys: "It is both a privilege and pleasure to present to the Academy Professor Sir Edwin Southern, or Ed as he would much prefer to be called. He really needs no introduction, because of his extraordinary accomplishments in molecular biology and genomics."

edit Introduction

Blotting methods same as saying: Methods of Nucleic acid analysis

Examination of DNA or RNA from particular gene. It requires to be able to distinguish specific DNA segments or RNA molecules.

DNA analysis

• Problem: find and examine the specific fragment, generated by restriction enzyme digestion

RNA analysis

• Problem: detect and measure the amount and quality of a particular mRNA transcript

The solution:

• Use of gel electrophoresis to separate the molecules of DNA or RNA by size
• Carrying out the nucleic acid hybridizatiion with a probe to identify the molecule of interest

edit Some important definitions for this topic

Blots are techniques for transferring DNA , RNA and proteins onto a carrier so they can be separated; often follows the use of a gel electrophoresis.

Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field.

edit Types of Blotting Methods

The main ones are:

• Southern blot: used to detedt DNA

• Nouthern blot: used to detect RNA

• Wertern blot: used to detect protein

But there are also these ones:

• Southwestern blot: involves identifying and characterizing DNA-binding proteins by their ability to bind to specific oligonucleotide probes (1980, B. Bowen and colleagues)

• Reverse Northern blot: for RNA; the nucleic acid immobilized on a membrane (collection of isolated DNA fragments rather than RNA), and the probe is RNA extracted from a tissue and radioactively labelled

• Far-Western blot: uses an antibody to detect a protein of interest

• Far-Eastern blot: for Lipids, Drugs and Hormones (1994 Tokyo); analysis of lipids separated by high-performance thin layer chromatography (HPTLC)

• Dot blot (or Slot): used to detect biomolecules

• Eastern blot: Used to analyze protein post translational modifications (PTM)

In some sources it is said that this method doesn’t exist but in others it is an important method

edit Southern blotting

Also called a DNA blot

Allows investigators to determine the molecular weight of a restriction fragment and to measure relative amounts in different samples.

Involves:

• Separation
• Transfer
• Hybridization

DNA detected can be:

• single gene
• or part of a larger piece of DNA (such as a viral genome)

edit Procedure

1 The DNA is digested with a restriction enzyme.

2 The complex mixture of fragments is subjected to gel electrophoresis to separate the fragments according to size.

3 The restriction fragments present in the gel are denatured with alkali

• • transferred onto a nitrocellulose filter or nylon membrane by blotting.

4 The filter is incubated under hybridization conditions with a specific radio-labeled DNA probe.

• • The probe hybridizes to the complementary DNA restriction fragment.

5 Excess probe is washed away and the probe bound to the filter is detected by autoradiography, which reveals the DNA fragment to which the probe hybridized.

This is an example of the result in the end of this procedure.

edit Northern blotting

Detection and quantitation of mRNA levels

Developed by James Alwine and George Stark at Stanford University

Was named such by analogy to Southern blotting

The preferred method:

• For determining transcript size

• For detecting alternatively spliced transcripts.

RNA samples are first separated by size

• via electrophoresis
  • In an agarose gel
  • under denaturing conditions

Includes:

• Transferation
• Cross-llinkage
• Hybridization with labeled probe

These can all be used as hybridization probes:

• radiolabeled or nonisotopically labeled DNA
• in vitro transcribed RNA
• Oligonucleotides

But there is some limitations. These are usually:

• if RNA samples are degraded à the quality of the data and the ability to quantitate expression are compromised.
• a standard procedure is less sensitive
• the difficulty associated with multiple probe analysis. To detect more than one message, it is usually necessary to strip the initial probe before hybridizing with a second probe. This process can be time consuming and problematic.

edit Procedure

This procedure is almost equal to that in Southern Blotting method.

The only differences are:

• It is the RNA which isolated from several biological samples (RNA is more susceptible to degradation than DNA)

• The labeled probe is detected via autoradiography or via a chemiluminescence reaction. In both cases this results in the formation of a dark band on an X-ray film.

edit Western blotting

Is an Immunoblotting technique

Rely on the specificity of binding between a molecule of interest and a probe.

• Molecule of interest: a protein

• Probe: an antibody raised against that particular protein

Gel electrophores applied

Separated by size.

Just like in the other blots: the result is blotted onto a special paper

Allow:

• determination of the molecular weight of a protein

• measurement of the relative amounts of protein present in different samples

edit Procedure

1 Proteins are separated by gel electrophoresis.

2 The proteins are transfered to a sheet of special blotting paper called nitrocellulose.

3 The proteins retain the same pattern of separation they had on the gel.

4 The blot is incubated with a generic protein to bind to any remaining sticky places on the nitrocellulose.

5 An antibody is then added to the solution which is able to bind to its specific protein.

• • The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or dye attached.

6 The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed.

edit Links

edit Related Articles

edit External Links

• http://www.molecularstation.com/dna/southern-blot/

• http://www.bio.davidson.edu/COURSES/GENOMICS/method/Southernblot.html

• http://www.med.unc.edu/pmbb/MBT2000/Northern%20Blotting.htm

• http://www.bme.gatech.edu/vcl/WesternBlotting/Background/Introduction.htm

• http://www.bio.davidson.edu/COURSES/GENOMICS/method/westernblot.html

• http://teachline.ls.huji.ac.il/72320/methods-tutorial/western/western-blot.html

• http://en.wikipedia.org/wiki/Electrophoresis

• http://ns1.web-books.com/MoBio/Free/Ch9D.htm

• http://en.wikipedia.org/wiki/Blot_%28biology%29

• http://www.ambion.com/techlib/basics/northerns/

• http://biobasics.gc.ca/english/View.asp?x=736#blotting

edit References

1. ↑ Incomplete citation of article.  . Honorary Fellow citation for Sir Edwin Southern. The Academy of Medical Sciences. 2009, y. 2009, vol. 1, p. 1,