Sunday, 2 March 2014

Genome mapping with Restriction Enzymes: Y10 Lambda Project Part II

The use of Restriction Enzymes (or to give them their proper name, Restriction Endonucleases) to selectively cut the genetic material, DNA, opened the way for screening patients for genetic diseases around 30 years ago. These enzymes are isolated from bacteria and naturally recognise and cut specific, short (4-8 base-pair), sequences of DNA. We have been generously supplied with a range of these enzymes by New England Biolabs for our lab project. The most "famous" Restriction Enzyme is probably EcoRI (the name is derived from the organism it is extracted from [E.coli] the strain [R] and the order in which it was discovered [I,II etc]). Hence the restriction enzyme BamHI comes from Bacillus amyloliquifaciens, strain H, and it was the first such enzyme to be isolated from this strain.

In the 1980s in particular, the combination of these enzymes and the use of radioactive phosphorus to detect minute quantities of DNA, made it possible to detect differences in the properties of genes in patients suspected of passing on genetic diseases during pregnancy. The technique for detecting the DNA fragments generated by these enzymes was known as Southern Blotting, after its inventor Professor Ed Southern (on the right there is an image of one such diagnostic blot). Under some circumstances, genetic mutations would give rise to changes in the restriction enzyme patterns, which would help doctors diagnose genetic diseases as a first step towards treatment.

Phage lambda DNA is a linear duplex of DNA, when isolated from the phage particles. We will use this DNA as a substrate for a set of restriction enzymes. The enzymes will be incubated with the DNA and the cleaved fragments run on an agarose gel. The banding patterns will be recorded and the sizes of the DNA fragments measured alongside some DNA fragments of known size (usually expressed in base pairs). The lambda genome can be analysed using free online software in order to determine the position of our restriction sites and the genes that control the development of the phage. In this way we shall combine experimental analysis with the use of Bioinformatics, just as Molecular Biologists do in their research work.

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