| Other methods for the detection of variation in DNA
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| Single-strand conformational polymorphism (SSCP)
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| SSCP is useful for analyzing PCR products of 200 bp and less and is moderately sensitive
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| TRINUCLEOTIDE REPEATS IN GENES GIVE RISE TO HUMAN DISEASE |
| A 33-year-old woman has recently been diagnosed as having myotonic dystrophy (MD). MD is characterized by autosomal dominant inheritance, muscular weakness associated with impaired muscular relaxation, frontal balding, endocrine disorders such as diabetes and premature ovarian failure, and premature cataract formation. The patient has muscular problems and frontal balding, whereas her affected father, aged 60 years, has only just developed cataracts. She is concerned about the risk to her own children if she becomes pregnant. |
| Comment. While the majority of nucleotide repeats are non-coding, several trinucleotide repeats have been described, which are related to human disease. The majority of trinucleotide repeats are clinically silent, but 11 clinical disorders have been identified where trinucleotide repeats appear directly responsible for a disease phenotype (see Fig. 34.16), and where the repeat is said to be unstable. Unstable repeats undergo changes in the size of the repeat during meiotic division and generally undergo expansion of the size of the repeat so that with each successive meiosis, i.e. from generation to generation, the number of repeats will gradually increase. Once trinucleotide expansions reach a critical size they begin to interfere with gene function and then result in the clinical syndrome (Table 34.6). In the case of myotonic dystrophy, with an expansion size of 80 repeats, the patient will exhibit the signs of the disease. Owing to the instability of the triplet repeat during meiosis, offspring of this patient might expect to have a larger expansion and also display features of the disease. Similarly, successive generations may display signs of MD at a younger age. This phenomenon of progressive worsening of the clinical phenotype with successive meioses is known as anticipation and is the result of progressive enlargement of the trinucleotide expansions during meiosis (Fig. 34.17). Interestingly, anticipation may be sex-limited, with the largest expansions only present in eggs, which have larger amounts of cytoplasm than spermatozoa. This observation has been suggested as the explanation for the occurrence of neonatal MD (a life-threatening form of the disease seen in newborn children) only in cases where the mother donates the triplet expansion to the offspring. |
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| Figure 34.16 Molecular diagnosis of Huntington's disease. This blot illustrates the use of a PCR-based method to diagnose Huntington's disease (HD). A portion of the HD gene, including the disease-associated triplet expansion region, is amplified by PCR. Each individual has two copies of the gene, one on each chromosome 4, and each PCR generates two PCR products. The PCR products are electrophoresized and compared with a size-specific ladder, which enables the size of the PCR product, and thus the number of triplet repeats in each product, to be assessed accurately. Affected patients will have triplet expansions greater than 35 repeats. HD is inherited as an autosomal-dominant trait, so that the disease is expressed on inheritance of a single copy of the mutant gene. |
| Single-stranded DNA has a tendency to form complex structures by folding back on itself, just as tRNA molecules form
hairpin structures. The mobility of single-stranded DNA in an electrophoretic gel depends not only on the length of the DNA but also on its conformation, which is determined by its DNA sequence and folding. Therefore, changes in DNA structure that do not alter the length of the target DNA, i.e. substitutions in contrast to deletions or insertions, can alter the mobility of the fragment in a nondenaturing gel, so-called single-strand conformational polymorphisms (SSCP). SSCP is
performed by PCR with a radioactive primer. The reaction products are denatured to render them single-stranded, and diluted to prevent intermolecular hybridization. The DNA is then electrophoresized on non-denaturing polyacrylamide gels. A control sample is also included on the gel to allow identification of the variant from the wild-type allele.
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