A mutation in a non-coding DNA sequence is believed to affect expression of the gene coding for a specific fetal enzyme. Liver and bone marrow cells from the fetus and his parents are obtained. Which of the following is the best method to determine if this gene is being transcribed in cultures of the isolated cells?

Question

A mutation in a non-coding DNA sequence is believed to affect expression of the gene coding for a specific fetal enzyme.  Liver and bone marrow cells from the fetus and his parents are obtained.  Which of the following is the best method to determine if this gene is being transcribed in cultures of the isolated cells?

Quizplus · Accepted Answer

11ec160b_3753_9b4a_9249_b90668b27f9f_MD0001_00 The Southern, Western, Northern and Southwestern blot procedures are techniques used to analyze and identify DNA fragments, proteins, mRNA, and DNA-bound proteins, respectively. The best method for determining whether a gene is undergoing transcription is to analyze for the presence of its mRNA using a Northern blot. In the case described above, Northern blot analysis of each of the cell culture samples can determine if mRNA corresponding to the gene of interest is being transcribed. All of the "blot" tests rely on the same basic techniques. First, the components of the unknown sample - DNA for Southern blots, mRNA for Northern blots, protein for Western blots, and DNA-bound protein for Southwestern blots - are separated by size and charge via gel electrophoresis. The resultant bands are then blotted onto a nitrocellulose membrane and incubated with a labeled hybridization probe or antibody to identify the specific DNA fragment, mRNA molecule or protein of interest. (Choice B) A complete failure of gene expression would lead to a failure to produce the protein, which can be detected by Western blotting. However, the question is asking how to determine if the gene is being transcribed (genes may be transcribed but not properly expressed if there is a problem with protein translation). As transcription refers to the production of mRNA from a DNA template, a test that detects specific mRNA sequences (Northern blot) would be most appropriate. (Choices C and D) This patient's mutation in a non-coding DNA sequence (likely affecting a promotor or enhancer region) is resulting in decreased gene expression. A Southern or Southwestern blot could potentially be used to detect the mutated DNA sequence and assess for transcription factor binding. However, the degree of transcription of the affected gene is better assessed through direct detection of its associated mRNA. (Choice E) The ELISA (enzyme-linked immunosorbent assay) is a test commonly employed to measure the amount of a protein in body fluids. It can be quantitative, for example, to measure plasma insulin levels. Educational objective: Northern blots detect target mRNA in a sample and can be used to assess the degree of gene transcription.

A Mutation in a Non-Coding DNA Sequence Is Believed to Affect