Third Generation DNA Sequencing with a Nanopore - Nanopores: Sensing and Fundamental Biological Interactions - page 299

Fig. 12.5 Stretching DNA in a nanopore. (a) qPCR results obtained for the 2 nm pores showing
the copy number vs. voltage. 622 bp dsDNA permeates the 2 nm pore in 10 nm thick Si 3 N 4
membrane ( top inset ) for V > 2.5 V , and the 1.8 2.2 nm pore in 20 nm thick Si 3 N 4 membrane

( bottom inset ) for V > 5.5 V .(b) qPCR results indicating the number of MS3 DNA copies that

permeate through the 1.8 0.2 nm pore shown in the inset as a function of the membrane voltage.

Insets on the left and right are snapshots of methylated and unmethylated MS3 translocating
through the 1.8 nm pore respectively. Both DNA exhibit an ordered B-DNA form, but there is a
significant degree of disorder for unmethylated DNA. The highlighted region of the strand shows
the portion of the DNA where methylated cytosines are located. The same region is also high-
lighted in the unmethylated strand for comparison. Adapted from references [ 48 , 49 , 59 ]

threshold for unmethylated MS3 in Fig. 12.5b is U ¼ 3.6 V while hemi- and fully

methylated MS3 show U ¼ 2.7 V, respectively.

The large shifts in the thresholds with methylation are surprising because the
leading nucleotides in the strand that are important to stretching in a pore are
separated by more than 18 bp (~6 nm) from a methylation site. The structure of
methylated DNA, inferred from X-ray diffraction and NMR, indicates that the
effect of methylation on the conformation of DNA is very subtle, and localized
near the methylation site [ 59 ]. On the other hand, MD simulations indicate that the
methyl groups reduce the DNA flexibility because of the steric hindrance by bulky
methyl groups and because the DNA folds around methyl groups [ 60 ]. Snapshots of

3.2 V and U ¼