Southern blot: Difference between revisions
No edit summary |
No edit summary |
||
| (7 intermediate revisions by 4 users not shown) | |||
| Line 1: | Line 1: | ||
Named after its originator [[Sir Professor Edwin Southern|Sir Professor Edwin Southern]]<ref>http://www.brookes.ac.uk/about/honorary/profiles/edwin_southern</ref>, Southern blot analysis is the separation by movement through a [[Non-denaturing gel|non-denaturing gel]] such as [[Agarose|agarose]] by the process of [[Electrophoresis|electrophoresis]]. Instead of [[Agarose|agarose]], the gel may also be [[Urea|urea]] or [[Polyacrylamide|polyacrylamide]] and the denaturation step is therefore not necessary. The sample is | Named after its originator [[Sir Professor Edwin Southern|Sir Professor Edwin Southern]]<ref>http://www.brookes.ac.uk/about/honorary/profiles/edwin_southern</ref>, Southern blot analysis is the separation by movement through a [[Non-denaturing gel|non-denaturing gel]] such as [[Agarose|agarose]] by the process of [[Electrophoresis|electrophoresis]]. Instead of [[Agarose|agarose]], the gel may also be [[Urea|urea]] or [[Polyacrylamide|polyacrylamide]] and the denaturation step is therefore not necessary. The sample is denatured using an [[Alkali|alkali]] such as [[Sodium hydroxide|sodium hydroxide]], which separates the double-stranded [[DNA|DNA]] into single strands<ref>Hartl D., Ruvolo M. (2012) Genetics: Analysis of Genes and Genomes, 8th Edition, Vermont: Jones and Bartlett Learning</ref>, and blotted with a sheet of [[Nitrocellulose|nitrocellulose]] or [[Nylon|nylon]]. It is important that the positions of the bands from the [[Electrophoresis|electrophoresis]] gel are conserved on the [[Nitrocellulose filter|nitrocellulose filter]]. This is achieved using [[Absorbent paper|absorbent paper]] - the paper is layered upon the nitrocellulose sheet and it absorbs the [[Water|water]] molecules from the gel up through the sheet, carrying the single strands of DNA with them. However, the DNA strands do not make it to the paper - they bind to the nitrocellulose filter instead, remaining within the bands they formed during electrophoresis. | ||
This blot is then hybridised with a labelled, radioactive probe of a [[DNA|DNA]] or [[RNA|RNA]] which possesses a complementary sequence to the target DNA. The filter is then washed to dispose of any unbound probe, and viewed using [[X-ray film|X-ray film]]. The bands of DNA which contain the labelled probe will appear as dark regions on the film - these are the fragments which have the desired sequence<ref>Hartl D., Ruvolo M. (2012) Genetics: Analysis of Genes and Genomes, 8th Edition, Vermont: Jones and Bartlett Learning</ref>. | |||
[[Alleles|Alleles]] in the sample are distinguishable by either the fragment sizes or the strength of hybridisation<ref>Bradley et al. Medical Genetics (3rd Ed)</ref>. | |||
=== References === | === References === | ||
<references /> | <references /> | ||
Latest revision as of 17:31, 22 October 2018
Named after its originator Sir Professor Edwin Southern[1], Southern blot analysis is the separation by movement through a non-denaturing gel such as agarose by the process of electrophoresis. Instead of agarose, the gel may also be urea or polyacrylamide and the denaturation step is therefore not necessary. The sample is denatured using an alkali such as sodium hydroxide, which separates the double-stranded DNA into single strands[2], and blotted with a sheet of nitrocellulose or nylon. It is important that the positions of the bands from the electrophoresis gel are conserved on the nitrocellulose filter. This is achieved using absorbent paper - the paper is layered upon the nitrocellulose sheet and it absorbs the water molecules from the gel up through the sheet, carrying the single strands of DNA with them. However, the DNA strands do not make it to the paper - they bind to the nitrocellulose filter instead, remaining within the bands they formed during electrophoresis.
This blot is then hybridised with a labelled, radioactive probe of a DNA or RNA which possesses a complementary sequence to the target DNA. The filter is then washed to dispose of any unbound probe, and viewed using X-ray film. The bands of DNA which contain the labelled probe will appear as dark regions on the film - these are the fragments which have the desired sequence[3].
Alleles in the sample are distinguishable by either the fragment sizes or the strength of hybridisation[4].
References
- ↑ http://www.brookes.ac.uk/about/honorary/profiles/edwin_southern
- ↑ Hartl D., Ruvolo M. (2012) Genetics: Analysis of Genes and Genomes, 8th Edition, Vermont: Jones and Bartlett Learning
- ↑ Hartl D., Ruvolo M. (2012) Genetics: Analysis of Genes and Genomes, 8th Edition, Vermont: Jones and Bartlett Learning
- ↑ Bradley et al. Medical Genetics (3rd Ed)