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. 2016 Sep 14;9(1-2):61-66.
doi: 10.3727/215517916X693087. eCollection 2017 Jan 8.

Measurement of DNA Length Changes Upon CpG Hypermethylation by Microfluidic Molecular Stretching

Daisuke Onoshima  1 , Naoko Kawakita  2 , Daiki Takeshita  2 , Hirohiko Niioka  3 , Hiroshi Yukawa  2 , Jun Miyake  3 , Yoshinobu Baba  4
Affiliations

Measurement of DNA Length Changes Upon CpG Hypermethylation by Microfluidic Molecular Stretching

Daisuke Onoshima et al. Cell Med. .

Abstract

Abnormal DNA methylation in CpG-rich promoters is recognized as a distinct molecular feature of precursor lesions to cancer. Such unintended methylation can occur during in vitro differentiation of stem cells. It takes place in a subset of genes during the differentiation or expansion of stem cell derivatives under general culture conditions, which may need to be monitored in future cell transplantation studies. Here we demonstrate a microfluidic device for investigating morphological length changes in DNA methylation. Arrayed polymer chains of single DNA molecules were fluorescently observed by parallel trapping and stretching in the microfluidic channel. This observational study revealed that the shortened DNA length is due to the increased rigidity of the methylated DNA molecule. The trapping rate of the device for DNA molecules was substantially unaffected by changes in the CpG methylation.

Keywords: Cytosine-guanine dinucleotides (CpG); DNA methylation; Microfluidic device; Single-molecule detection.

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Figures

Figure 1
Figure 1
Schematic of the single-molecule length measurement of methylated DNA through DNA stretching by the microfluidic device.
Figure 2
Figure 2
Detailed explanation of the microchip and aligned single DNA molecules on zigzag lines for length measurement.
Figure 3
Figure 3
Results for nonmethylated (A) 48.5-kbp DNA and (B) 17-kbp DNA samples. The histograms of the length measurements and fluorescence images of single DNA molecules are shown. Scale bar: 10 μm.
Figure 4
Figure 4
Results for methylated 48.5-kbp DNA. (A) The histogram of length measurements and a fluorescence image of a single DNA molecule. Scale bar: 10 μm. (B) Gel electrophoresis result. (1) Nonmethylated 48.5-kbp DNA, (2) methylated 48.5-kbp DNA with BstUI, (3) nonmethylated 48.5-kbp DNA with BstUI, and (4) a DNA ladder are shown.
Figure 5
Figure 5
Trapping rate analysis of nonmethylated and methylated DNA.
See this image and copyright information in PMC

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