This Data Analysis Problem does not appear in the textbook.

Source: Lu, S., P. J. A. Davies. 1997. Regulation of the expression of the tissue transglutaminase gene by DNA methylation. Proc. Natl. Acad. Sci. USA 94: 4692–4697.

Corresponding chapter(s) in the textbook: Chapter 4

Review the following terms before working on the problem: DNA methylation, restriction endonucleases, modification methylases, agarose gel electrophoresis, ethidium bromide, UV box

Experiment

This experiment studied the effect of DNA methylation on restriction endonuclease cleavage. Equal amounts of a 1.74 kb human DNA fragment were either methylated using bacterial modification methylase HpaII (samples 1–3) or left unmethylated (samples 4–6). Aliquots of these DNA samples were then incubated without enzyme (samples 1 and 4), with the restriction endonuclease MspI (samples 2 and 5), or with the restriction endonuclease HpaII (samples 3 and 6). Lanes labeled M contain size markers. DNA fragments were separated by agarose gel electrophoresis. The gel was stained with ethidium bromide and photographed on a UV box.

Figure

© Copyright (1997) National Academy of Sciences, U.S.A.

Questions

1. How many MspI and HpaII cleavage sites are present in the 1.74 kb DNA fragment (compare samples 4, 5, and 6)?

2. Why do MspI and HpaII produce identical band patterns (sample 5 and 6)?

3. How does methylation affect DNA cleavage by HpaII (compare samples 3 and 6)?

4. How does methylation affect DNA cleavage by MspI (compare samples 2 and 5)?

5. Suggest an explanation for the differences in the products of MspI and HpaII in this experiment.