Convenient continuous sonication method for the preparation of medium-sized polydeoxynucleotides

Abstract

We recently reported results on polydeoxynucleotides prepared for 31p NMR studies by sonication [1]. For these studies we required samples of medium length, from ca. 50-200 base pairs (bp), comparable to the size of DNA in nucleosome core particles. To obtain this size range we utilized the continuous low-temperature sonication procedure described for calf-thymus DNA by Davis and Phillips [2]. We have extended this procedure to small volumes of synthetic polydeoxynucleotides in a convenient preparative method. The apparatus used is shown in Fig. 1. The temperature was continuously monitored and kept between 0-4°C using a circulating cooling bath. Volumes down to 1 ml are possible in this apparatus, although we generally used 2-4 ml containing 5-100 A260n m units in 0.1-1.0M NaCI solution. A tapered microprobe was used with a He~t Systems-Ultrasonics W225R sonicator. The power level was optimized just below observable cavitation-bubbling (ca. 25 W). Experiments comparing the efficiency of breakage using the pulsed capability for comparable time periods (e.g. 50% duty cycle for twice the total time) indicated that continuous sonication was more efficient. The size distribution of the samples after sonication was determined by gel electrophoresis. Following development of the gel in ethidium bromide, the photographic negatives were scanned with a microdensitometer (Joyce-Loebl double beam, model IIIB) and the scans were digitized. The distances migrated were converted to base pairs relative to known standards using the formula described by Kovacic and Van Holde [3]. Results for sonication for different time periods showed that it was possible to obtain fairly well defined distributions with several base sequences [1]. For example, 10 min sonication of poly(dGdC), poly(dGdC) gave a maximum at