MULTIPLEX DISPLAY POLYMERASE CHAIN-REACTION AMPLIFIES AND RESOLVES RELATED SEQUENCES SHARING A SINGLE MODERATELY CONSERVED DOMAIN

We present a technique, multiplex display polymerase chain reaction (M D-PCR), that amplifies and resolves coding sequences from messenger RN As sharing only a single moderately conserved domain encoding eight or nine amino acids. The technique, a form of single-sided PCR, allows d etection of known and novel genes in a family by suing one degenerate primer complementary to a gene family-specific domain. A second common primer is complementary to an oligonucleotide ligated to a nearby res triction enzyme cleavage site. Uniquely, restriction enzyme digestion of single-stranded cDNA, a technique never previously performed to use ful advantage, is used to increase the specificity and sensitivity of the technique. Up to several hundred bases of coding sequence are ampl ified simultaneously from many (potentially from all) genes in a speci fic family, yielding products of different sizes from different genes, and allowing amplified products to be resolved electrophoretically. T ypically, more than 50% of the amplified sequences are from the target ed gene family and many of the amplified products are novel sequences. mRNAs representing less than 1 in 100,000 messages can be detected. T he method allows the focused yet open-ended examination of genes in fa milies known to be important in both normal cellular homeostasis and t he etiology of many diseases. (C) 1998 Academic Press.