Homework09 BIS101/Engebrecht In 8th edition: Chapter 11: 17, 21, 22, 29, 31, 34, 35; Chapter 12: 32, 35, 36 In 9th edition: Chapter 20: 16, 18, 21, 22 1. How would you go about cloning the yeast HIS4 gene? Assume that you have a yeast his4 mutant and a genomic library that can replicate in yeast available but no sequence information. Clone gene by complementation of the mutant phenotype. To do this, transform mutant with genomic library and select for cells that can grow on medium lacking histidine. These should contain the wild-type copy of the gene that is complementing the mutant. 2. You successfully cloned the gene. You now need to analyze the DNA. Given the following information, please draw a circular restriction map of the vector and DNA fragment that represents the gene. The 5 kb vector has a single BamHI site that was used to construct the library. The vector also has a single EcoRI site but has no HindIII sites. Digestion of your DNA with BamHI generates 2 fragments: 3.5kb, 5kb Digestion of your DNA with EcoRI generates 3 fragments: 1kb, 2kb, 5.5kb Digestion of your DNA with HindIII generates 1 fragment: 8.5kb Digestion of your DNA with both BamHI and EcoRI generates 5 fragments: 0.5kb, 1kb, 1kb, 1.5kb, 4.5kb Digestion of your DNA with both EcoRI and HindIII generates 4 fragments: 0.7kb, 1kb, 2kb, 4.8kb Digestion of your DNA with both HindIII and BamHI generates 3 fragments: 0.3kb, 3.2kb,
5.0kb
3. Consider the following pedigrees. Assume all people marrying into the pedigree do not carry the abnormal allele.
I3
I1
I2
I4
II 1
II 2
II 3
II 4
II 5
II 6
III 1
III 2
III 3
III 4
III 5
III 6
III 7
a. What is the most likely mode of inheritance of this disease? Autosomal because recessive affected children from unaffected parents, few affected children.
b. If individuals II2 and II4 have a child, what is the probability that the child will have the disease? 2/3 x 1 x 1/4 = 1/6 c. If individuals III4 and III5 have a child, what is the probability that the child will have the disease? 2/3 x 1/2 x 1/2 x 1/4 = 1/24 d. VNTR analysis is performed with two different primer pairs (1 and 2) on all individuals in the pedigrees above with the following results: I1 12 500bp 400bp 300bp 200bp 100bp I2 12 I3 12 I4 12 II1 12 II2 12 II3 12 II4 12 II5 12 II6 12 -
III1 12 500bp 400bp 300bp 200bp 100bp
III2 12 -
III3 12 -
III4 12 -
III5 12 -
III6 12 -
III7 12 -
Which primer pair, and which band is associated with the disease locus? Primer pair 1; 100bp band
e. Given these results, please reassess the probability of II2 and II4 having a child with the disease and the probability of III4 and III5 having a child with the disease (assume very tight linkage with the VNTR). The probability of II2 and II4 having a child with the disease would be most likely 1/4 since we know that both are heterozygous for the associated VNTR; the probability of III4 and III5 having a child with the disease is most likely 0 since III5 does not carry the associated VNTR.
4. Please write an exam question for the final (it can be on any of the material we have covered throughout the course). Please include the corresponding answer. I will use at least 1 (possibly more) of these questions in the final exam. If you are not enrolled in a BIS101D section you may turn the question/answer in directly to me, either in class or via e-mail. I will accept questions until Friday, June 4.