The ẞ-globin gene codes for the ẞ-globin polypeptide of haemoglobin. It has two alleles, HbA (normal) and Hbs (sickle cell). The sickle cell allele differs from the normal allele due to a base substitution mutation and this mutation results in a single amino acid change to the ẞ-globin polypeptide. There are three possible genotypes and phenotypes. • Hbs Hbs, sickle cell anaemia, a severe disease • HbA Hbs, sickle cell trait with mild or no symptoms of sickle cell anaemia • HbA HbA, normal (healthy) A man and woman who both have sickle cell trait may choose to have children by IVF. This allows the genotype of embryos to be determined by gene testing before the embryos are implanted. Embryos with the normal genotype can then be selected and implanted into the mother. One technique that can be used in gene testing an embryo for the Hbs allele is restriction fragment length polymorphism (RFLP) analysis. This involves digesting a DNA sample from an embryo with a restriction endonuclease and then separating the DNA fragments by gel electrophoresis. The position of the DNA fragments on the gel can show if the embryo has the Hbs allele.

1. (a)(i)Name the technique used to produce many copies of a DNA sequence from a very small quantity of DNA.[1]
2. (a)(ii)Explain why it is necessary to copy this DNA sequence many times in order to test embryos for Hbs alleles by RFLP analysis.[1]
3. (b)With reference to Fig. 3.1, Fig. 3.2 and Fig. 3.3, explain why the enzyme MstII can be used in RFLP analysis to show the difference between these parts of the HbA and Hbs alleles.[4]
4. (c)After cutting with MstII, the DNA fragments are separated by gel electrophoresis. Explain how gel electrophoresis separates DNA fragments cut with restriction endonucleases.[3]
5. (d)(i)State the purpose of using DNA from individuals homozygous for HbA and for Hbs.[1]
6. (d)(ii)With reference to Fig. 3.4, complete Table 3.1 to show the genotypes of embryos 2, 3 and 4. Table 3.1 embryo genotype 1 HbA HbA 2 3 4 [2]
7. (e)Discuss the ethical and social considerations of gene testing embryos for genetic diseases.[3]

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