BIOL 443 Molecular BiologyAssignment for Analysis of a mutant Hemoglobin using restriction mapping

DUE DATE: Monday, December 5, 2011, 5:00 PM

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The figure above shows bands that should have been produced by digestion of normal Rabbit β globin DNA with EcoRI and Hae III. The gel to the right shows the results of digests of normal (N) and mutant (M) Rabbit β globin DNA with EcoRI and Hae III.

1. Fill in the table with the Rabbit β globin fragment sizes from gel lanes 2 and 4. Lane 4 possess plasmid

fragments (2)

Lane 1 2 3 4

Fragmentsizes

4.1 4.1 1.4 1.2

2.5 N/aA

1.2 <0.8

0.8 N/a 0.9 <0.7

2. Compare the sizes and numbers of fragments in the EcoRI digests of the normal and mutant Rabbit β globin.

Describe your observation (2) Theoretically, the mutant Rabbit beta globin are missing one or two beta alleles from normal hemoglobin. Point mutations that causes thalassemia can block digestion of EcoRI, which causes the effect of less fragments, and therefore, the number of fragments of the mutant Rabbit beta globin are less. The mutant Rabbit beta globin is missing fragments 2.5 and 0.8. For some reason, it appears that the missing fragments may be too large to travel therefore it might be arrested at the top where the white tracking dye is at in lane 2. Fragment 2.5 and 0.8 was not cut right by EcoRI causes the two fragment to appear larger in size. EcoRI possess less recognition sequences than Hae III.

3. Compare the sizes and numbers of fragments in the Hae III digests of the normal and mutant Rabbit β globin. Describe your observation (2)

Hae III possess more recognition sequences than EcoRI, therefore, the numbers of fragments are more than EcoRI. Since Hae III create more fragments it affects the size of the Rabbit beta globin also by decreasing it into smaller fragments. The mutant Rabbit beta globin however, is missing fragment 1.4 and 0.9 in lane 4. Looking closely into the fragment sizes, there is a second 0.9 fragment that is supposed to be stacked on top of fragment 0.9 in the normal Hb in lane 3. In this order respectively, these three fragments 1.4, 0.9, and 0.9 are closely together in the mutant allele that is upstream. Conclusion: the Hae III combined fragments of 1.4, 0.9, and 0.9 from the mutant Rabbit beta globin could be larger on some fragments than the normal hemoglobin. Point mutations that causes thalassemia can block digestion of Hae III, which causes the effect of less fragments, and therefore, some of the fragments of the mutant Rabbit beta globin are larger. Another important observation on Hae III is that this restriction enzyme can help remove the plasmid fragments after fragment size 4.1 is isolated.

4. Based on your observations, what type of mutation is in the mutant Rabbit β globin. (2)

Based on this analysis from one of the two major groups of beta globin mutation, this mutation can be categorized as a nondeletion form. In the nondeletion form group, the affected area is near upstream and also the promoter region before the beta globin gene. Based on observations, the mutation on the Rabbit beta globin could be a point mutation which is a type of substitution within the nondeletion form. This mutation could not be a single insertion or deletion because it will cause a frame shift that will affect everything that is downstream. However, It could be one or multiple insertions and deletions that cancel out each other e.g. (+ + , - -). In Hae III observations, the fragments affected by the mutation are 1.4, 0.9, and 0.9 are upstream. The downstream fragments are still able to with hold their recognition sequences and are properly targeted by restriction enzymes.

5. Give the name of the disease that is caused by this type of mutation in the β globin gene in human DNA. (1)The name of this disease caused by this type of mutation in the Beta globin gene in human DNA is called Beta Thalassemia.