The word 'mutation' derives from the Latin 'mutatio' meaning change or alteration (Marshall 2002). In the context of genetics, the term 'mutation' refers to a 'permanent structural change in the DNA' (www.genome.gov/glossary) (Cotton and Scriver 1998) and may involve changes in individual base pairs of DNA or larger structural changes in DNA. In some contexts and disciplines, 'mutation' has become associated with a 'disease-causing change' in the DNA, notably in terms of clinical genetics, while in the public perception there has been an increasingly negative association with the term 'mutation' (Condit et al. 2002). By contrast 'polymorphism' was regarded as a 'non-disease-causing change' or 'neutral' DNA variant (Cotton and Scriver 1998). The term polymorphism is used to refer to 'common variation in the sequence of DNA among individuals' (www.genome.gov/glossary) and more specifically to refer to sequence variants present at a frequency of 1% or more in a population, without reference to a particular phenotypic effect (Cotton and Scriver 1998).
A further question in terms of terminology when describing DNA sequence variants relates to the terms 'mutation' or 'polymorphism' (Box 1.14). Sequence variants at their outset involve a de novo mutational event in the DNA of a single individual, and in general the term mutation is used in the context of a rare variant causing disease, but variants ('mutations') may rise to high allele frequencies in particular populations as seen with Hb S. Moreover, while the vast majority of common sequence variants described as 'polymorphisms' have no functional consequence, some are recognized to play an important role in common multifactorial diseases. When considering fine scale sequence changes or diversity, the phrase 'DNA sequence variant' carries no assumption about disease association or frequency. The occurrence of the variant should be validated and carefully assigned in terms of location and nature using specific terminology and unique identifiers as discussed.
1.2.4 Methods of detecting the Hb S DNA sequence variant
A restriction fragment length polymorphism (RFLP)
was first found for a specific site 5 kb downstream of the HBB gene recognized by the restriction enzyme Hpal, which showed variation in the length of the digested restriction fragments between individuals, specifically among those of African descent in whom additional bands (7 and 13 kb in length) were recognized in addition to the expected size of 7.6 kb (Kan and Dozy 1978). Significantly, the 13 kb bands were associated with individuals who had sickle haemoglobin. Kan and Dozy noted in their paper that this 'may be useful for the prediction of the sickle cell gene in prenatal diagnosis' and that 'polymorphism in a restriction enzyme site could be considered as a new class of genetic marker and may offer a new approach to linkage analysis and anthropological studies' -views that were to be substantially validated although more specific restriction sites for the Hb S variant and other methods of detection were to develop.
Restriction sites spanning the specific single nucleotide variant were utilized to discriminate between DNA sequence for the two alleles, for example the enzymes Ddel or Mstll (Geever et al. 1981; Chang and Kan 1982). In the presence of the A to T substitution the recognition site is lost and the enzyme no longer cuts, allowing those individuals with sickle cell trait and sickle cell disease to be identified (Fig. 1.17). Initial work relied on Southern blotting to detect DNA sequences. In this technique, invented by Edwin Southern, restriction enzyme digested DNA is separated on the basis of size by agarose gel electrophoresis, transferred to a membrane and then hybridized with a labelled (usually radioactive) probe to enable detection (Southern 1975).
The development of the polymerase chain reaction (PCR) made this process significantly faster and easier, with very much less DNA required (Fig. 1.18) (Mullis and Faloona 1987). One of the earliest applications of PCR was to detect the Hb S DNA sequence variant. DNA was amplified by PCR using the Klenow fragment of
Hb S sequence variant
Restriction site for MstII (CCTNAGG)
template strand non-template
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