Problem Set 12 - Genetics 380 : Spring 2011 Problem Set 12...

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Unformatted text preview: Genetics 380 : Spring 2011 Problem Set 12 (Answers due the week of 4/18) 1. A linear molecule of DNA is digested with the following combinations of enzymes: Enzymes BamHI BamHI + Hind III Hind III Fragments (kb) 2.5 1.5 1.8 1.3 1.0 1.5 0.7 0.8 1.2 0.7 0.5 a. How many BamHI and HindIII sites are there in the DNA molecule? a. Draw a restriction map of the molecule. 2. A plasmid is digested with the following combinations of enzymes, yielding the following bands on an agarose gel. Enzymes EcoRI Fragments (kb) 7.8 EcoRI + SalI 4.7 SalI 4.7 SalI + BamHI 2.5 1.4 2.1 2.2 1.0 1.0 2.1 0.7 1.0 a. How many EcoRI, SalI and BamHI sites are there in the plasmid? a. Draw a restriction map of the plasmid. BamHI 7.8 BamHI + EcoRI 3.9 3. A circular plasmid has a single HindIII restriction enzyme site in the middle of a tetracycliner resistance gene (tet ). Drosophila genomic DNA is digested with HindIII, and a library is made in the plasmid. Probing reveals that clone 15 contains a specific Drosophila gene of interest. Clone 15 is studied by restriction analysis with HindIII and another enzyme EcoRV. The ethidium bromide stained electrophoretic gel shows bands as illustrated in the diagram below (the control was the plasmid without the insert DNA). The sizes of bands (in kilobases) are shown alongside. Assume that all bands in the gel are linear molecules. a. Draw restriction maps for the plasmid with and without the insert, showing the sites of r the target sequences and the approximate position of the tet gene. a. If the same tet r gene cloned in a completely non-homologous vector is made radioactive and used as a probe in a Southern blot of this gel, which bands do you expect to appear radioactive on an autoradiogram? 4. DNA sequencing is performed by the dideoxy chain-termination method on a cloned piece of DNA. A part of the autoradiogram of the sequencing gel is given below. 5. DNA studies are performed on a large family that shows a certain autosomal dominant disease of late onset (approximately 40 years of age). A DNA sample from each family member is digested with the restriction enzyme TaqI and subjected to gel electrophoresis. A Southern blot is then performed, with the use of a radioactive probe consisting of a part of human DNA cloned in a bacterial plasmid. The autoradiogram, aligned with the family pedigree, is as follows. Affected (diseased) members are shown in black. 5 kb 3 kb 2 kb a. b. What is the relationship between the DNA variation, the probe DNA, and the gene for the disease? Draw the relevant chromosome regions. How do you explain the last son? 6. The gene for the autosomal dominant disease shown in this pedigree is thought to be on chromosome 4, so five RFLPs (1-5) mapped on chromosome 4 were tested in all family members. The results are shown in the diagram; the superscripts represent different alleles of the 5 RFLP loci. a. Decide which RFLP locus is closest to the disease gene (explain your logic). b. How would you use this information to clone the disease gene? ...
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