Multiplex Display Polymerase Chain Reaction Amplifies and Resolves Related Sequences Sharing a Single Moderately Conserved Domain Franklin V. Peale, Jr.,* ,1 Karen Mason,† Andrew W. Hunter,† and Mark Bothwell† *Department of Pathology and †Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195 Received August 28, 1997 We present a technique, multiplex display polymer- ase chain reaction (MD-PCR), that amplifies and re- solves coding sequences from messenger RNAs shar- ing only a single moderately conserved domain encoding eight or nine amino acids. The technique, a form of single-sided PCR, allows detection of known and novel genes in a family by using one degenerate primer complementary to a gene family-specific do- main. A second common primer is complementary to an oligonucleotide ligated to a nearby restriction en- zyme cleavage site. Uniquely, restriction enzyme di- gestion of single-stranded cDNA, a technique never previously performed to useful advantage, is used to increase the specificity and sensitivity of the tech- nique. Up to several hundred bases of coding sequence are amplified simultaneously from many (potentially from all) genes in a specific family, yielding products of different sizes from different genes, and allowing amplified products to be resolved electrophoretically. Typically, more than 50% of the amplified sequences are from the targeted gene family and many of the amplified products are novel sequences. mRNAs rep- resenting less than 1 in 100,000 messages can be de- tected. The method allows the focused yet open-ended examination of genes in families known to be impor- tant in both normal cellular homeostasis and the eti- ology of many diseases. © 1998 Academic Press The large number and variety of techniques devised to identify genes that are characteristically expressed in a tissue, including subtractive hybridization (1), comprehensive sequencing of cDNAs (2), reverse Northern blotting (3), differential display PCR (4), rep- resentational difference analysis (5, 6), serial analysis of gene expression (7, 8), and oligonucleotide array hybridization (9), reflect the potential value of the in- formation to be obtained. For several of these tech- niques (subtractive hybridization and differential dis- play PCR are notable examples), initial excitement and widespread application were followed by a backlash of disappointment as their limitations became apparent. However, all approaches have particular strengths and weaknesses: techniques suited to quantifying the ex- pression of low-abundance mRNAs (oligonucleotide ar- ray hybridization or reverse Northern blotting) are limited to studying previously cloned genes; con- versely, methods capable of identifying novel genes, including differential display and subtractive hybrid- ization, may not efficiently detect rare sequences (10 – 12). Comprehensive sequencing is limited by the fact that most sequences, including those derived from crit- ical cell regulatory genes, are present in low abundance in an mRNA population (13). Therefore, while a high proportion of randomly selected cDNAs represent rare transcripts, identification of any specific gene ex- pressed at low abundance cannot be accomplished without redundant sequence analysis of the 1–3  10 4 genes expressed in a tissue (8, 12). One of the most powerful and widely applied tech- niques has been the polymerase chain reaction used with degenerate primers, which can amplify multiple sequences, both known and novel, from specific gene families (14, 15). However, this strategy requires the various target genes to share two regions of nucleotide homology so that both sense and anti-sense degenerate PCR primers can be designed; divergent genes lacking homology in either region will not be amplified. A fur- ther limitation is that the products derived from re- lated genes are typically all the same length, hindering rapid determination of the number and sequence of the 1 To whom correspondence should be addressed. Fax: (206) 543- 0934. E-mail: fpeale@u.washington.edu. 158 0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. ANALYTICAL BIOCHEMISTRY 256, 158 –168 (1998) ARTICLE NO. AB972500