AFM characterization of single strand-specific endonuclease activity on linear DNA

Abstract

The specificity of nucleases for nicked and un-nicked double-stranded DNA has been characterized using atomic force microscopy (AFM). We have found that AFM has advantages over the usual macroscopic analyses, such as sucrose gradient centrifugation or electrophoresis, in characterizing nuclease digestion. In particular, short DNA fragments resulting from non-specific digestion were detected and, thus, the true length distribution of digested DNA was revealed. A simple numerical method is proposed to estimate the number of nicked sites per DNA molecule based on AFM images.

Figure 1

AFM images of pBR322 DNA purified by gel electrophoresis in low melting point agarose. (a and b) Supercoiled and nicked/relaxed circular forms, respectively. (c and d) Linear DNAs prepared by ScaI digestion from DNA in (a) and (b), respectively. Magnified images of individual DNA molecules are presented as insets in (a) and (b).

Figure 2

Agarose gel electrophoresis of linear DNA digested with S1 nuclease. Lanes 1 and 8, molecular weight marker (λ DNA digested with EcoRI and HindIII); lanes 2–7, linear DNA prepared from supercoiled pBR322; lanes 9–14, linear DNA prepared from nicked pBR322. S1 nuclease was added at 0, 0.6, 1.8, 3.6, 18 and 36 U in lanes 2–7 and 9–14, respectively.

Figure 3

AFM images and the corresponding length distributions of S1-treated un-nicked (Ia–Id) and nicked (IIe–IIh) DNA. S1 nuclease was added at 0.6, 1.8, 18 and 36 U in (a)–(d) and (e)–(h), respectively. The arrows in (c) indicate the digested DNA. The number of DNA molecules was plotted against the measured DNA length (left, short; right, full-length, 1.4 µm) in a histogram presentation.

Figure 4

Digestion of linear DNA by mung bean nuclease. The DNA sample and amount of mung bean nuclease added were as follows: (a) un-nicked, 2 U; (b) un-nicked, 20 U; (c) nicked, 2 U; (d) nicked, 20 U. The amount of DNA was 0.15 µg in all cases. The number of DNA molecules was plotted against the measured DNA length (left, short; right, full-length, 1.4 µm).

Figure 5

AFM images and the corresponding histograms of S1 digestion of (a) unirradiated control DNA and (b) UV-irradiated DNA. Twenty units of S1 nuclease were reacted with 0.15 µg of DNA. The number of DNA molecules was plotted against the measured DNA length (left, short; right, full-length, 0.3 µm) in the histograms.