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BIOL 1003
Elizabeth Nisbet

Genetic screening detects particular genes or chromosome mutations (e.g. cystic fibrosis, …)  DNA extraction (e.g. white blood cells, gametes)  Cut the DNA at gene loci with restriction enzymes  Split DNA fragments up on the basis of their size with electrophoresis gel  Southern blotting and use of radioactive DNA probe to locate the fragments of DNA  Autoradiography to create an image of the DNA pattern Stage 1 - DNA extraction  Small sample of tissue (e.g. blood) is mixed with water-saturated phenol and chloroform  Causes proteins to precipitate out leaving DNA in the water layer  DNA can now be extracted from the water layer and purified Stage 2 - Restriction enzymes  Each restriction enzyme is specific to one base sequence  Cut the DNA (cleavage) after enzymes have attached to all recognition sites  Fragments produced are called restriction fragment length polymorphisms (RFLPs)  Some produce blunt ends, some sticky ends (more useful) Stage 3 - Electrophoresis  Electrophoresis separates DNA fragments according to their size and electrical charge  DNA mixture is placed in a well at one end of a gel (made of agarose)  Electrical current will move the DNA fragments to the positively charged electrode  Phosphate is highly positive, making nucleotide negative Stage 4 - Southern Blotting and DNA probes  Heat DNA on the gel to unwind and make single stranded DNA  A nylon membrane placed over the gel is covered with absorbent paper / single stranded fragments are transferred to membrane by capillary action  Fix fragments on membrane with UV light  Put membrane into solution containing the DNA probe  DNA probe attaches to complementary base sequences of the disease-causing gene / fragment is labelled radioactive Stage 5 - Autoradiography  Radioactive solution is washed off and an X-Ray plate is placed over the membrane  Radioactive probes (32p) will give off radiation causing a pattern of bands on the X-ray plate, conforming the presence of the disease causing gene  Mutant gene is missing a restriction site which
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