Application of Kinetic Polymerase Chain Reaction and Molecular Beacon Assays to Pooled Analyses and High- Throughput Genotyping for Candidate Genes Min Shi, 1 Diana Caprau, 1 John Dagle, 1 Lene Christiansen, 2 Kaare Christensen, 2 Jeffrey C. Murray 1 * 1 Department of Pediatrics, University of Iowa, Iowa City, Iowa 2 Epidemiology, Institute of Public Health, University of Southern Denmark, Odense University, Odense, Denmark Received 23 January 2003; Accepted 29 October 2003 BACKGROUND: The addition of DNA analysis to epidemiologic studies that have traditionally incorporated demographic and interview data can provide additional power and open new avenues for investigation. DNA can be obtained from a variety of tissues, but each has attendant variation in sample quantity, quality, and cost of acquisition. Analytic approaches for DNA genotyping are under constant development, but current appli- cations allow small amounts (less than 2 ng per assay) of DNA to be used for genotyping. METHODS: In this report, we designed effective assays for a spectrum of genes using either kinetic polymerase chain reaction (PCR) or molecular beacon applications. We also investigated the extent to which DNA use and reagent cost could be minimized. Kinetic PCR assays were also applied to investigate the potential of pooled sample analysis. RESULTS: Our results show that small amounts of DNA can be successfully amplified in a high- throughput fashion using both kinetic PCR and molecular beacon methods. Greater than 97% of the genotype results from these two methods are consistent. In addition, error rates in allele frequency measurements using DNA pools of 100 or more samples were often less than 1% and usually less than 3%, which provides another option for substantially minimizing the costs of genotyping in studies involving large numbers of individuals. CONCLUSIONS: Effective assays have been designed for a spectrum of genes widely studied in birth defects, including: MTHFR, NAT1, TGFA, RFC1, PAX9, EPHX1, and SKI. An efficient assay has been designed for the detection of the presence of X and Y chromosomes, which can be applied to the studies of sex chromosome abnormalities or sample quality control. Birth Defects Research (Part A) 70:65–74, 2004. © 2004 Wiley-Liss, Inc. INTRODUCTION For studies involving large numbers of individuals, power increases with the numbers of samples studied. However, increasing the number of samples also increases cost. When DNA is included in this analysis, the cost of genotyping can quickly become substantial in studies that might potentially benefit from the analysis of dozens, or even hundreds, of individual genes or loci. In the last two decades, a movement from Southern-blot– based restric- tion fragment length polymorphism (RFLP) analyses to polymerase chain reaction (PCR)– based analyses has of- fered the promise that thousands of samples can be reliably typed for genetic variation at hundreds of loci in a cost- effective manner. In addition, an alternative to individual genotyping is the use of pooled sample analysis, which was first introduced by Arnheim et al. (1985) and has had varying success in frequency determinations for individual alleles, as well as in linkage studies, since that time (Arn- heim et al., 1985; Murray et al., 1987; Carmi et al., 1995; Scott et al., 1996; Barcellos et al., 1997; Germer et al., 2000; Zhou et al., 2001). We report here our experience with identifying cost-effective assays using both kinetic PCR (Germer et al., 2000; Chen et al., 2002) and molecular beacons (Tyagi et al., 1998; Marras et al., 1999) on a quan- titative PCR apparatus for a variety of candidate genes valuable for studies of birth defects and other disorders. The kinetic PCR assay was also evaluated in pooled sample analyses. We also included comparisons of DNA samples Grant sponsor: National Institutes of Health; Grant numbers: R01 DE11948, DE08559, P60 DE13076-02; Grant sponsor: Centers for Disease Control and Prevention; Grant number: U50/CCU 71328. *Correspondence to: Jeffrey C. Murray, M.D., University of Iowa, 140 Eckstein Medical Research Building, Iowa City, IA 52242. E-mail: jeff-murray@uiowa.edu DOI: 10.1002/bdra.10153 © 2004 Wiley-Liss, Inc. Birth Defects Research (Part A) 70:65–74 (2004) Birth Defects Research (Part A): Clinical and Molecular Teratology 70:65–74 (2004)