- DNA probes are used to diagnose diseases caused by defective genes or oncogenes
- Single stranded complementary DNA strand (cDNA) is labelled with an radioactive isotope
- Need to know specific base sequence found in defective gene
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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
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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
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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)
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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
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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
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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
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Using enzymes to diagnose disease |
| Detecting the body's own 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
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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
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