Nucleic Acids Research, 2005, Vol. 33, No. 10 e91 doi:10.1093/nar/gni089 Genome amplification of single sperm using multiple displacement amplification Zhengwen Jiang 1 , Xingqi Zhang 2 , Ranjan Deka1 and Li Jin3,1,* 1Department of Environmental Health, Center for Genome Information, University of Cincinnati College of Medicine, 3223 Eden Ave, Cincinnati, OH 45267, USA, 2Department of Obstetrics and Gynecology, Northwestern University Medical School, Chicago, IL, USA and 3State Key Laboratory of Genetic Engineering and Center for Anthropological Studies, School of Life Sciences and Morgan-Tan International Center for Life Sciences, Fudan University, Shanghai, China Received February 2, 2005; Revised April 11, 2005; Accepted May 17, 2005 ABSTRACT Sperm typing is an effective way to study recombina- tion rate on a fine scale in regions of interest. There are two strategies for the amplification of single mei- otic recombinants: repulsion-phase allele-specific PCR and whole genome amplification (WGA). The former can selectively amplify single recombinant molecules from a batch of sperm but is not scalable for high-throughput operation. Currently, primer extension pre-amplification is the only method used in WGA of single sperm, whereas it has limited capa- city to produce high-coverage products enough for the analysis of local recombination rate in multiple large regions. Here, we applied for the first time a recently developed WGA method, multiple displace- ment amplification (MDA), to amplify single sperm DNA, and demonstrated its great potential for produ- cing high-yield and high-coverage products. In a 50 ml reaction, 76 or 93% of loci can be amplified at least 2500- or 250-fold, respectively, from single sperm DNA, and second-round MDA can further offer >200- fold amplification. The MDA products are usable for a variety of genetic applications, including sequencing and microsatellite marker and single nucleotide poly- morphism (SNP) analysis. The use of MDA in single sperm amplification may open a new era for studies on local recombination rates. INTRODUCTION A detailed knowledge of linkage disequilibrium (LD) patterns across the human genome was widely considered a prerequisite for comprehensive association testing (1). Recent data have shown that LD in human populations is highly structured into discrete blocks with limited haplotype diversity (2–5). This LD structure was believed to result from the inter- play between recombination hotspots (3,5,6) and the demo- graphic history of human populations (7,8). Little is known about the role of recombination in shaping LD patterns in populations, although statistical approaches may provide some clues (9–11). The answer to this question may lie in compar- ison of population LD structure with the distribution of meiotic crossovers. Sperm typing can identify the distribution of male local meiotic recombination rate, which can at least partially explain the LD pattern, as exempliﬁed by Jeffreys et al. (3). Two strategies have been taken for detecting highly local- ized meiotic recombination hotspots in sperm. One is to amplify single recombinant molecules using repulsion-phase allele-speciﬁc PCR from a large batch of sperm DNA, fol- lowed by localization of crossover sites (3,12–14). The other is to scan thousands of single sperm cells to identify and localize meiotic recombinants (15–17). The former is efﬁcient for ﬁne mapping of crossover sites in a deﬁned region of several kilo- bases without laborious work on screening recombinants as required in the latter. However, it can only be used in studies on small regions of <10 kb and the workers have to be very careful of possible contamination from artiﬁcial recombinants resulting from template switching during PCR ampliﬁcation (18,19). For the second method, single sperm cells were ﬁrst pre-ampliﬁed by either multiple PCR or whole genome amp- liﬁcation (WGA) to produce sufﬁcient DNA for further mul- tiple genotyping reactions to identify and localize meiotic recombinants. Although multiple PCR has been more widely used for single sperm analysis (20–22), WGA seems to be more promising and preferred for ﬁne mapping of meiotic recombination sites because it can amplify many more marker loci than multiple PCR. Primer extension pre-ampliﬁcation (PEP) was developed to amplify single sperm DNA on *To whom correspondence should be addressed at Liren Biology Building, Room 220, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China. 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