Method for Rapid Restriction Analysis of YAC Clones

The development of the yeast artificial chromosome (YACs) technology (3) has provided, by virtue of its capacity to carry large fragments of genomic DNA, the possibility of linking the genetic map and the physical map of an organism. This requires the alignment of overlapping YAC clones into contigs for which a number of strategies have been described as follows: the use of sequence tagged sites (STSs) (8), YAC fingerprinting using dispersed repeat sequences (2) and the use of rare cutter enzyme mapping (9). A method for YAC alignment by partial restriction analysis has recently been described (10). However, this method is not very reproducible in our hands, since no or very little digestion was observed with 100 units of enzyme and an incubation of one hour at the appropriate temperature. To overcome this problem, a series of modifications of this latter method (essentially by replacing bovine serum albumin with casein and decreasing the amount of restriction enzyme), enabled us to obtain a highly flexible protocol for the rapid restriction mapping of YAC clones. Our method is based on partial digestion of YACs with rare cutter enzymes and pulsed-field gel electrophoresis (4). Agarose blocks, from individual YAC clones, were prepared (1) and washed overnight in 1× TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) at 4°C to remove the excess EDTA. Partial digestion of YACs by time limitation was performed in a total volume of 200 μL, which was obtained by making the following mixture: 20 μL of 10× casein mixture [i.e., 1 mg/mL casein (6), 20 μM dithiothreitol (DTT) and 40 μM spermidine]; 20 μL of the appropriate 10× concentrated reaction buffer (supplied with the enzyme), 80 μL ddH2O and 80 μL of YAC containing agarose block. This mixture was cooled on ice for 30 min, and the appropriate amount of enzyme was subsequently added. The mixture was again incubated on ice for 30 min to allow diffusion of the enzyme in the agarose block. Following these incubation steps, the mixture was transferred to a water bath to bring it to the temperature recommended by the manufacturer of the enzyme. Digestion was terminated through the addition of 1 mL of 0.5 M EDTA, pH 8.0. Fragments were separated on a 1% pulsedfield gel, blotted onto Hybond-N membranes (Amersham, Arlington Heights, IL, USA), hybridized, washed and autoradiographed as previously described (5). A 2.7-kb pBR322 PvuII/BamHI fragment was used to detect the left arm (ARS1/TRP1), and a 1.7-kb PvuII/ BamHI fragment was used to detect the right arm (URA3). Figure 1 shows the results from these experiments by using different amounts of MluI on a 250-kb sugar beet YAC clone. This figure shows that, depending on the amount of enzyme used, partial digestion is accomplished between 5 and 10 min when 10 U of enzyme are used (Panel A), or between 5 and 30 min when 5 U of enzyme are used (Panel B). These results also show that, by using 5 U of enzyme together with the casein containing reaction mixture, the incubation time is less critical, resulting in a flexible method, which is highly reproducible. Repeating this experiment in the absence of the casein mixture resulted in no or very little digestion of the agarose-embedded YAC DNA after 1 h of incubation at the appropriate temperature. Further investigations showed that other enzymes like NotI, NarI and EagI (37°C digestion) had the same range of partial digestion when 5 U of these enzymes were used, while enzymes like SfiI and BssHII (50°C digestion) showed complete partial digestion between 5 and 20 min. These optimized conditions enabled us to map several YAC clones with 5 different restriction enzymes, using only one lane of a pulsed-field gel per YAC and restriction enzyme. Our method allows the construction of restriction maps of YAC clones in a rapid, very reproducible and economical (in time and resources) way and will therefore be of use for rapid alignment of YAC clones into contigs.