The measurement of exonuclease activities by atomic force microscopy |
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Authors: | K Hori T Takahashi T Okada |
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Institution: | (1) Joint Research Center for Atom Technology, c/o National Institute for Advanced Interdisciplinary Research, 1-1-4, Higashi, Tsukuba, Ibaraki 305, Japan, JP |
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Abstract: | We have applied atomic force microscopy (AFM) to the measurement of BAL 31 nuclease activities. BAL 31 nuclease, a species
of exonuclease, is used to remove unwanted sequences from the termini of DNA before cloning. For cutting out only the appropriate
sequences, it is important to know the nuclease properties, such as digestion speed and the distribution of the lengths of
the digested DNA. AFM was used to obtain accurate measurements on the lengths of DNA fragments before and after BAL 31 nuclease
digestion. We analyzed 4 DNAs with known number of base pairs (288, 778, 1818, and 3162 base pairs) for correlating the contour
length measured by AFM with the number of base pairs under the deposition conditions used. We used this calibration for analyzing
DNA degradation by BAL 31 nuclease from the AFM measurement of contour lengths of digested DNAs. In addition, the distribution
of digested DNA could be analyzed in more detail by AFM than by electrophoresis, because digested DNA were measured as a population
by electrophoresis, but were measured individually by AFM. These results show that AFM will be a useful new technique for
measuring nuclease activities.
Received: 8 August 1997 / Accepted: 10 September 1997 |
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Keywords: | DNA Enzyme activity Atomic force microscopy Exonuclease BAL 31 nuclease |
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