Accurate length determination of DNA molecules visualized by atomic force microscopy: evidence for a partial B- to A-form transition on mica

Achieving the most correct estimate of the contour length of digitized DNA molecules is a key aspect of the microscopic analysis of nucleic acids by e ither electron microscopy (EM) or atomic force microscopy (AFM). Six differ ent methods, that are mathematically not too complex and suited for common, practical use, have been tested here using simulated polymers in two dimen sions and real DNA molecules (564, 1054, 2049 and 4297 bp long) imaged in a ir by AFM. The main result is that the frequently used Freeman estimator (L -F = n(e) + root 2n(o)) overestimates the real contour length of the polyme rs by about 4%. More accurate estimates are obtained with the Kulpa estimat or (L-K = 0.948n(e) + 1.343n(o)) or with the corner count estimator (L-C = 0.980n(e) + 1.406n(o) - 0.091n(e)). In the range of the DNA sizes and magni fications we have considered, however, the best results are obtained with a n ad hoc developed routine that smoothes the DNA trace by a polynomial fitt ing of degree 3 over a moving window of 5 points. Under these conditions, t he difference between the measured and the real contour length of the molec ules is less than 0.4% . The accuracy of this procedure allowed us to revea l a discrete, size-dependent, shortening of DNA molecules deposited onto mi ca under low salt conditions and imaged in air by AFM. Awareness of this st ructural alteration, that can be attributed to a partial transition from B- to A-form DNA, may lead to a more correct interpretation of DNA molecules or protein-DNA complexes imaged by AFM. (C) 2001 Elsevier Science B.V. All rights reserved.