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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