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(Questions 9-11)Allele specific oligonucleotides (ASO) can be used to determine the different alleles present in a DNA sample. Single sperm (from a single donor), which have completed meiosis, can be used for a polymerase chain reaction (PCR). The amplified DNA is then spotted to filters and hybridized with probes that discriminate between single nucleotide substitutions. Four primers (called A, B, D, and E) were used to amplify DNA samples and ASO probes were used to determine the allele present, visualized as a black dot. For each locus, the left side of the filter is probed with one ASO (e.g. A1) and the right side is probed with the other one (e.g. A2). (5.0K)
(Questions 12 and 13) Microsatellites are very useful as DNA markers. They consist of 1-3 bp units repeated 15-100 times in a row. They are highly polymorphic, with multiple alleles that differ in 2 or 3 bp unit increments. The multiple alleles can be detected as PCR products of different sizes. One particular microsatellite has four alleles in the population and is closely linked to an autosomal dominant form of early Alzheimer’s disease.
(Questions 18-20)Huntington disease (HD) is a lethal neurodegenerative disorder that exhibits autosomal dominant inheritance. Because the onset of symptoms is usually not until the third, fourth, or fifth decade of life, patients with HD usually have already had their children, and some of them inherit the disease. Now that the gene for HD has been identified and cloned, a direct genetic test can be performed to inform people in an HD family of their genetic status. The mutation that causes HD is an expansion of CAG repeats within the coding sequence of the gene. The normal allele contains up to 34 repeats, while disease-causing alleles carry 42 or more repeats. These repeats always expand, never contract. In a particular HD family, a young woman in her early 20s has a mother whose brother is exhibiting symptoms of HD. The mother is 38 years old, has not been tested for HD and is not currently exhibiting any symptoms. There is no history of HD on the father’s side of the family.
(Questions 22-25)You are using positional cloning to identify a gene involved in hereditary breast cancer. You have access to a large family with many members that have developed breast cancer at an early age.