Table 6. Genotypes of seven SNPs in genomic DNA, eight sperm aliquots and the second-round MDA product of S16 determined by Taqman technology PAGE 8 OF 9 rs1323690 G/A ? G A A G A G A A e91 Nucleic Acids Research, 2005, Vol. 33, No. 10 Samples Genomic DNA S04 S05 S06 S08 S13 S14 S15 S16 S16MDA2 ACE rs4575595 G/A G ? G G A G ? G G rs11868324 rs4267385 G/A A ? A A G A ? A A T/C T C T T C T ? T T ? indicates no amplification. method utilizes the F29 mesophilic polymerase for primer extension. The F29 polymerase has strong strand displace- ment activity and is able to extend >70 kb on DNA templates. Even stable secondary structure in the template does not affect the processivity and strand displacement ability of the enzyme (39). Polymerase slippage was demonstrated to include four major steps: (i) arrest of DNA synthesis within direct repeats, (ii) dissociation of the polymerase from the template, (iii) unpairing of the 30 end of the newly synthesized strand and its reannealing to the neighboring repeat and (iv) resumption of DNA synthesis (37). The aforementioned features of F29 polymerase, which prevent the dissociation of the enzyme from the template, are therefore theoretically able to drastic- ally reduce the rate of slippage at microsatellite sites when F29 polymerase is used, in contrast to other polymerases without strand displacement activity. Hellani et al. (26) has observed no microsatellite slippage at 16 loci in MDA pro- ducts of 40 single leukocytes. Our data also showed that all alleles at ﬁve STR sites detected in MDA products of eight sperm aliquots were identical to parental alleles in genomic DNA, indicating no microsatellite slippage occurred. It was, therefore, demonstrated that MDA products from single sperm cells were useful for further microsatellite analysis. From the genotyping data of single sperm cells, we can easily determine the two haplotypes in the sample studied. For example, haplotypes composed of rs6129731, rs2235362 and rs6102287 should be AAA and CCG (Table 4), and for the ACE gene they are GAT and AGC (Table 6). Currently, almost all available haplotypes with a high density of SNPs in human population have been inferred by statistical methods from phase-unknown genotypes in random samples or trio families (see data from PGA, EGP and HapMap projects). Further LD analysis was performed based on the inferred haplotype data. Little is known about how well these inferred haplotypes match the true ones, and how much inference error can affect the LD pattern. It is necessary to investigate some represent- ative regions by comparing experimentally determined true haplotypes with those inferred statistically. Here, we demon- strated that MDA in single sperm cells can produce sufﬁcient DNA for thousands of genotyping reactions. By genotyping DNA from leukocytes and MDA products from one or two single sperm cells, the true haplotypes in a large chromosome region can therefore be determined. In this study, we found that sperm lysis at 65 C instead of on ice, as described in the manual supplied, could provide much better ampliﬁcation. It is possible that warm incubation is EDN1 rs5369 G/A G A G A A A G A A rs1800541 T/G T G T G G G T G G ELF5 rs1535721 G/A ? A G G A G A G G more efﬁcient in releasing DNA from the chromosomes. Except for alkaline lysis, some other studies also used pro- teinase K to treat cells before subjecting them to MDA (26,34). Rook et al. (26) tried alkaline lysis on ice and proteinase K digestion of laser capture-microdissected tissues, and decreased allele imbalance was observed for proteinase K treatment. Using proteinase K for lysis of sperm cells may, therefore, also produce good ampliﬁcation. We have demonstrated that MDA is an effective way for global genome ampliﬁcation in single sperm cells and can provide sufﬁcient single sperm DNA for genotyping in large quantity. It also makes possible ﬁne mapping of male meiotic recombination sites on a <10 kb level, and experi- mental determination of haplotypes in a large chromosome region, or even a whole chromosome. These maps can unam- biguously help better understand LD patterns in human popu- lation, thus further facilitating association studies. It can also improve our knowledge about the mechanism of recombina- tion, especially how recombination is initiated. ACKNOWLEDGEMENTS The authors thank Dr C. Stuart Baxter, Ming Dong, Ge Zhang, Huifeng Xi and all anonymous reviewers for helpful comments and Joanna Watson for help in STR and SNP genotyping. The authors also thank Dr Marian Miller for her help on figure edition. 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