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7 - The sickling disease page 5
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3. Diagnosis and prevention Link to the Medical Research Council web site
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Detecting the defective allele
Currently, prenatal diagnosis is the most widely used approach for the prevention of sickle cell anaemia. Chorionic villus sampling (CVS) allows prenatal diagnosis as early as eight weeks into a pregnancy. Fetal cells are obtained from the chorionic villi and the DNA is extracted and purified. Detection of the sickle cell allele involves the use of restriction enzymes, such as Hpal or EcoRl, which cut DNA into fragments at specific sequences of nucleotides (restriction sites). The resultant DNA restriction fragments are separated by electrophoresis.

The sickle cell mutation has the effect of altering one of the restriction sites adjacent to the b-globin allele. As a result, digestion of chromosomes with Hpal gives fragments of different lengths, depending on whether the normal haemoglobin allele (A) or the sickle cell allele (S) is present (see Figure 5).

Figure 6

Figure 5. Formation of restriction fragments from chromosomes containing (a) the normal b-globin allele (b) the sickle cell allele.

Electrophoresis is used to separate the DNA fragments on the basis of size, small fragments migrating further along a gel than large fragments. The separated fragments are then denatured with sodium hydroxide and transferred from the fragile gel by blotting onto a tough nitro-cellulose filter paper. The position of the DNA fragments is established using a radioactive gene probe. This is a sequence of radioactively labelled nucleotides complementary to the b-globin gene. When a solution containing the probe is washed over the nitro-cellulose filter, the probe will bind where it meets the complementary nucleotide sequence present in both the normal and mutant restriction fragments.

Questions 5 and 6
5. a) What is meant by: i) radioactive labelling; ii) complementary pairing?

b) What types of bond form between the nucleotides in the probe and the nucleotides in the restriction fragments?

c) How could you detect where the probe binds on the nitro-cellulose filter?

 

6. DNA purified from the blood of four individuals was analysed in this way. The electrophoresis results are shown in Figure 6.

Which of the results a-d corresponds to the following phenotypes:

i) sickle cell anaemia;
ii) normal haemoglobin;
iii) carrier?

Figures 7

Figures 6. Electrophoresis results from four individuals