Activity: Diagnosis of Huntington’s Disease
This exercise is designed to be a bridge between basic science & technology (DNA sequencing) and the applications of that technology (genetic testing). The purpose of this exercise is to size HD and non-HD alleles using the protocol used by the HD clinical testing laboratory at the UW Medical Center.
Materials needed: autoradiogram, calculator.
Understanding the autoradiogram: Orient it correctly; the lanes should be at the top.
The first four lanes on this gel constitute a sequencing ladder size control. The identity of this DNA is not relevant (maybe viral? bacterial?) -- it is only being used as a ladder, in which each band differs by only one nucleotide from the band above or below it.
The lanes marked 1-5 are DNA samples from 5 unrelated individuals. Each of these people came to UW to have the HD predictive test. They each contributed blood samples, from which DNA was isolated and subjected to a technique called polymerase chain reaction (PCR) to amplify the CAG repeat section of the HD gene on chromosome 4 (please refer to Fig. 4 of the HD Background Information handout.). The PCR products were spread out by polyacrylamide gel electrophoresis. Is it clear to you that four of the five individuals whose DNA appears on the autoradiogram have inherited two different alleles for the HD gene?
Diagnosing HD. DO CALCULATIONS IN YOUR JOURNAL, & SHOW YOUR WORK!
1. Begin with person #1. In his/her lane on this gel, you see two dark, one-inch wide smears. Each smear consists of many bands. The reason there is more than one band per smear, it turns out, is because the number of CAG repeats varies a little from cell to cell!
2. Find and use the highest, clearly discernable band within patient #1’s lower smear, and estimate how large it is (in base pairs) by looking at the sequencing ladder size control directly to the left. Note that several positions on the ladder (142 bp, 155 bp, etc.) have been marked for your convenience. Did you get about 144 bp?
3. Subtract 110 (this removes the nucleotides that were amplified but which were NOT part of the CAG repeats. Thus, 144 - 110 = 34 bp.
4. Divide by 3, since CAG is a trinucleotide repeat. 34 / 3 is about 11. Eleven CAG’s is well within the normal range of 10-34. See the bar graph from your HD Background Information handout. Thus, this allele for patient # 1 is normal.
5. Repeat steps 1-4 for patient #1’s other allele. If the number of CAG repeats in the second allele is also less than 39, you can diagnose this individual as having a negative HD test and she or he will most likely NOT develop Huntington’s disease.
6. Now repeat the analysis for the other four patients on this autoradiogram! It is difficult to read the sequencing ladder for patient # 4’s upper smear. Do the best you can. In reality,
what would technicians do?