Question

You wish to determine if there is a mutation somewhere within the promoter of the adult b-globin gene that could possibly be responsible for a case of b-thalassemia. Which ONE method, when compared to the same process performed on wild type DNA, would NOT provide you with information that could be consistent with this idea?

A) Prepare a pair of 18 nucleotide long primers that hybridize upstream and downstream of the promoter, perform PCR, and sequence the resulting fragment

B) Cut the DNA with a restriction enzyme that has sites both upstream and downstream of the promoter, perform a Southern blot on gel electrophoresis separated DNA fragments, hybridize to a 32P-labelled synthetic fragment from the region of interest, and obtain an autoradiograph of the membrane

C) Isolate DNA from the promoter region as in A) above, ligate it to a test gene sequence X, and test for the amount of X mRNA produced when the construct is transduced into a red blood cell line

D) Test the binding of known b-globin transcription factors to promoter DNA

I know that the correct answer is B, but I am not sure why. I would appreciate an explanation.

Answer 1

The critical step as it is given option B is the hybridization of the 32P-labelled synthetic fragment with the mutant adult beta globulin gene. Since the gene is mutated depending on the size and type of mutation the gene would have lost homologous regions with the 32P-labelled synthetic fragment and fail to hybridize. Even if the scale of mutation is small or there is rearrangemnent of the gene the fragment size of the hybridized DNA will vary and hence would not provided the desired result.