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Southern blotting

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DNA segments may be identified by the difference between base pairs on a pair of chromosomes. These differences may be as minimal as one base pair in five hundred but are sufficient to provide a restriction enzyme with an altered site for DNA cleavage. Consequently, new lengths of DNA fragments of different composition are produced after digestion.

The fragments are separated on the basis of molecular weight by agarose gel electrophoresis. The DNA is then denatured to give single stranded molecules. These are transferred to nitrocellulose paper which binds DNA. The position of single stranded DNA molecules on the blotting paper co-incides exactly with their original position on the gel. Next, a fully-characterized gene probe is used to bind to complementary regions of interest. The pattern that the probe takes up, as revealed by contrast techniques e.g. autoradiography, indicates the DNA segment which the gene of interest is located within. When the probe consists of complementary DNA to denatured DNA, the entire process is termed Southern blotting. Techniques also exist for using probes to RNA - Northern blotting - and protein - Western blotting.

The presence of distinct fragments can be used in conjunction with a family tree to establish which part of a chromosome carries a deliterious gene. The fragment of DNA is being used as a marker for genes in its locality that have manifest in a family's genotype.


General Practice Notebook
General Practice Notebook
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