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 is present at normal genes Mutant gene will travel shorter distances than normal DNA Using enzymes to diagnose pancreatitis Pancreas is found under the stomach and produces Hormones to regulate blood sugar levels and Digestive enzymes like amylase, lipase and trypsin that break down starch, lipids, and proteins respectively Insulin-dependent diabetics are unable to secrete insulin from pancreatic cells Pancreatitis is a disease of the pancreas Trypsin becomes active before released from the pancreas Pancreas is made of proteins Trypsin is active and digests/hydrolyses proteins Cell wall breaks down, amylase escapes into the blood Results of Successful Treatment Trypsin not activated early/enters the gut/does not enter blood Higher levels of trypsin in faeces since it passes through the gut (unaffected) Acute pancreatitis occurs suddenly. Diagnosed by the presence of amylase/lipase Chronic pancreatitis is a long-term condition Pancreas gradually loses its ability to produce enzymes There are low levels of pancreatic enzymes in faeces Fats pass through the intestine without being digested / fat is present in the faeces Using enzymes in biosensors Biosensors are made up of 2 enzymes and a colourless hydrogen-donor fixed on a strip The strip is dipped into a test solution (urine) Colour develops which indicates that glucose is present This method is used by diabetics to monitor their blood glucose levels Biosensors are easier than Benedict's reagent in detecting reducing sugars because biosensors work with two enzymes: glucose oxidase and perioxidase Glucose oxidase Highly sensitive to low conc. of glucose Highly specific because it only reacts with one specific substrate (glucose) Catalyses the conversion of glucose to hydrogen peroxide (H2O2) Peroxidase Catalyzes reaction between colourless hydrogen-donor molecule and H2O2 A coloured molecule is formed