A SIMULATION OF SUBTRACTIVE HYBRIDIZATION

Various strategies employed in genomic DNA cloning by subtractive hybr idization have been examined by computer simulations, with the compari son between the predictions and the published results, The result show s that the efficiency of target sequence enrichment and the sensitivit y to experimental conditions depend strongly on the enrichment strateg y employed, The strategy selecting only tester/tester after hybridizat ion can be very efficient to enrich targets. For successful target enr ichment, however, the strategy requires a highly efficient subtraction method and proper hybridization conditions, The strategy also require s 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 strateg y, the tester DNA selected may or may not be amplified by PCR before t he next round, In the case of the strategy selecting single-stranded t ester, the target DNA can be successfully enriched only when the selec ted DNA is directly used without PCR amplification in the next round. The strong features of existing methods can be combined to develop a p rotocol that is more efficient and more reliable.