AFM characterization of single strand-specific endonuclease activity on linear DNA.
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.
Umemura
K
K
Joint Research Center for Atom Technology (JRCAT), 1-1-4 Higashi, Tsukuba, Ibaraki 305-0046, Japan.
Nagami
F
F
Okada
T
T
Kuroda
R
R
D004247:DNA / Q000378:metabolism* / Q000648:ultrastructure; D004270:DNA, Circular / Q000378:metabolism / Q000648:ultrastructure; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D018625:Microscopy, Atomic Force; D010957:Plasmids / Q000648:ultrastructure; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*; D013379:Substrate Specificity
D004270:DNA, Circular; D004247:DNA; C047020:endodeoxyribonuclease ScaI; D015252:Deoxyribonucleases, Type II Site-Specific; D015719:Single-Strand Specific DNA and RNA Endonucleases