A simulation of subtractive hybridization.

Various strategies employed in genomic DNA cloning by subtractive hybridization have been examined by computer simulations, with the comparison between the predictions and the published results. The result shows that the efficiency of target sequence enrichment and the sensitivity to experimental conditions depend strongly on the enrichment strategy employed. The strategy selecting only tester/tester after hybridization can be very efficient to enrich targets. For successful target enrichment, however, the strategy requires a highly efficient subtraction method and proper hybridization conditions. The strategy also requires that the selected DNA be amplified by polymerase chain reaction (PCR) after each or each alternate subtraction. By contrast, the strategy selecting tester/tester plus single-stranded tester is less sensitive to various experimental factors, compared with the strategy selecting only tester/tester. However, it is not as efficient. With this strategy, the tester DNA selected may or may not be amplified by PCR before the next round. In the case of the strategy selecting single-stranded tester, the target DNA can be successfully enriched only when the selected DNA is directly used without PCR amplification in the next round. The strong features of existing methods can be combined to develop a protocol that is more efficient and more reliable.