Anchor Polymerase Chain Reaction Display: A High- Throughput Method to Resolve, Score, and Isolate Dimorphic Genetic Markers Based on Interspersed Repetitive DNA Elements Srinivas Ayyadevara,* , † John J. Thaden,† , ‡ and Robert J. Shmookler Reis* , † , ‡ , § ,1 *Department of Biochemistry and Molecular Biology, †Department of Geriatrics, and ‡Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and §Central Arkansas Veterans Health Care System, Little Rock, Arkansas 72205 Received January 7, 2000 Genes which confer a disease when mutated, or for which population variability contributes to a quanti- tative trait such as longevity or disease susceptibility, can be localized in the genetic map by use of an appro- priately dense set of polymorphic DNA markers. Here we describe an anchor PCR method for high-through- put genotyping, which can be used to amplify the DNA segments flanking an interspersed repetitive se- quence such as a transposon, and to limit the number of product bands per reaction to facilitate marker res- olution. We used this method to amplify and display DNA fragments flanking the Tc1 transposable ele- ments from different strains of the nematode Caeno- rhabditis elegans, varying widely in insert number, and to analyze marker segregation in recombinant inbred lines generated from an interstrain cross. Since essentially all eukaryotic genomes contain abundant interspersed repeat families, many of which are di- morphic (for presence or absence of specific elements) among populations, this method can be used for rapid genotyping and fine-scale chromosomal mapping in many species, including those for which extensive mapping and sequencing data do not yet exist. © 2000 Academic Press Key Words: genetic map; repetitive DNA; transposon; Tc1; C. elegans; polymorphic marker; quantitative trait locus. Identification of genes which govern a variable trait, or for which mutations cause or predispose to a disease, normally involves fine-scale genotyping of partial or entire genomes from many individuals, recombinant inbred lines, and/or congenic (back- crossed) lines. The power and resolution of genetic mapping depend on the sample size and the density of polymorphic markers used. Genotyping many in- dividuals or lines is labor-intensive, and is usually the rate-limiting step in linkage and association studies. We describe a new, high-throughput tech- nique capable of generating dimorphic markers at moderate to high density as required, without prior genomic sequencing, which can then be rapidly posi- tioned by genetic linkage. These markers can be amplified and scored very efficiently, at 30 –70 per reaction or lane, thus greatly facilitating mapping and positional cloning of genes governing diverse traits. Several methods based on polymerase chain reac- tion (PCR) have been used to amplify genomic DNA fragments flanking a specific known DNA sequence (1– 8). One of the strategies is anchor-ligation PCR, which comprises a number of variant procedures (e.g., 1–3) for amplification of DNA segments whose sequence is specified at only one end. Each method is accomplished in three steps: digestion of DNA with a restriction enzyme, ligation of an anchor sequence to the digested DNA, and then PCR amplification of anchor-ligated DNA with a site-specific primer and an anchor primer. Partial mismatch between the two anchor strands effectively prevents amplification of DNA fragments with anchor ligated to both ends 1 To whom correspondence should be addressed at J.L. McClellan Veterans Medical Center, Research-151, 4300 West 7th Street, Little Rock, AR 72205. Fax: 501-257-5578. E-mail: ReisRobertJS@ exchange.uams.edu. 0003-2697/00 $35.00 19 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Analytical Biochemistry 284, 19 –28 (2000) doi:10.1006/abio.2000.4636, available online at http://www.idealibrary.com on