GENETIC DISEASES

Gene Mutations

• Deletion, reading frame shifts • Substitution, one base

replaced by another • Duplication, repetition of part

of the sequence • Addition, Addition extra base • Change in one or more

nucleotide bases in the DNA • Change in the genotype (may

be inherited)

Cystic fibrosis

• People with cystic fibrosis inherit a defective gene on chromosome 7 called CFTR (cystic fibrosis transmembrane conductance regulator). Mutation causes the deletion of 3 bases in DNA resulting in one amino acid (phenylalanine) not being coded for

• The protein produced by this gene normally helps salt (sodium chloride) move in and out of cells. If the protein doesn't work correctly, that movement is blocked and an abnormally thick sticky mucous is produced on the outside of the cell.

• The cells most seriously affected by this are the lung cells. This mucous clogs the airways in the lungs, and increases the risk of infection by bacteria.

• The thick mucous also blocks ducts in the pancreas, so digestive enzymes can't get into the intestines. Without these enzymes, the intestines cannot properly digest food. People who have the disorder often do not get the nutrition they need to grow normally.

Cystic Fibrosis - Defective Gene

• Mutation causes the deletion of 3 bases in DNA. One amino acid (phenylalanine) is not coded for in theCystic Fibrosis Transmembrane Regulator CFTR protein

• Faulty CFTR protein cannot control the opening of chloride channels in the cell membrane

• Results in production of thick sticky mucus, especially in lungs, pancreas and liver

• Organs cannot function normally and infection rate increases

These are bacteria, Pseudomonas aeruginosa, a human pathogen that

is a particular problem in the lungs of cystic fibrosis patients

Phenylketonuria (PKU) – Defective Gene

• NORMAL – Melanin pigment produced

PKU

• Anna with her children Madeleine (centre), who has PKU, and Isobel. Madeleine's PKU is managed successfully by diet.

www.sciencemuseum.org.uk/exhibitions/genes

PKU

1. Gene mutation in DNA coding for the enzyme phenylalanine hydroxylase

2. Phenylalanine hydroxylase not produced

3. Amino acid phenylalanine cannot be converted to the amino acid tyrosine

4. Tyrosine is necessary to produce the pigment melanin

5. Phenylalanine collects in the blood and causes retardation in young children

6. Managed by controlling diet to eliminate proteins containing phenylalanine

7. Disease is tested by drops of blood taken from the baby

Genetic screening and DNA probes

• Genetic screening detects particular genes or chromosome mutations (e.g. cystic fibrosis, …)

• DNA probes are used to diagnose diseases caused by defective genes or oncogenes

• Need to know specific base sequence found in defective gene

Summary• 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)

Single stranded complementary DNA strand (cDNA) is labelled with an radioactive isotope

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

Southern blot• The first process of the Southern blot is agarose gel electrophoresis, a technique that

separates DNA fragments by size. • A mixture of DNA is digested with restriction enzymes, which cut the DNA into small

pieces of various sizes, and placed into different lanes of an agarose gel. • An electric current, which is run through the gel, forces the DNA fragments to move

through the agarose matrix. Next, the size-fractionated DNA, while still on the gel, is chemically denatured and blotted onto a sheet of nitrocellulose paper.

• The nitrocellulose is then heated so that the single stranded DNA fragments are permanently adhered to its surface. The Southern blot is now ready to be analyzed.

• A radioactive single stranded probe is hybridized with the DNA in question. The probe anneals to the DNA wherever a complementary sequence of genetic code exists.

• After the radioactive probe has been allowed to bond with the denatured DNA on the nitrocellulose paper, an X-ray is taken of the nitrocellulose sheet. Only the areas where the probe binds to the denatured DNA will show up on the X-ray film. This allows researchers to identify the spatial location and frequency of the specific genetic sequence contained by the probe (Brinton and Lieberman, 1994).