e91 Nucleic Acids Research, 2005, Vol. 33, No. 10 Table 3. Amplification rate for nine genes after second-round MDA treatment Genes used for quantification analysis Total copy number S16MDAa S16MDA2b Amplification rate TOP1 0.5 0 0 P53 5 2500 500 PAGE 6 OF 9 CYP1A1 PIK3CA C6orf195 ADCYAP1 PTEN PMS2 CAT 68 995 10 320 000 150 0.5 250 500 840 100 000 119 1225 202 500 165 1155 295 000 255 905 182 500 202 0 0 /c aThe input template DNA for the second-round MDA reaction, which is 1 ml of 1/10C0 MDA product of a single sperm aliquot S16. The copy number for each gene in the input DNA was determined by the total copy number of these genes in MDA products of S16 divided by 500. bThe total 50 ml second-round MDA product of S16. cUnable to calculate. Table 4. Genotypes of genomic DNA and nine sperm aliquots at five SNP loci determined by sequencing SNP ID rs6129731 rs2235362 rs6102287 rs1048943 rs17861120 Contigs NT_011362.9 NT_011362.9 NT_011362.9 NT_010194.16 NT_010194.16 4713602 4797334 4797565 45803303 45817497 A/C A/C A/G T/C A/G Position in contigs Genomic DNA S01 ? C G C G S04 A ? ? C G S05 A A A T A S06 A A A C/t A/g S08 C C G T A S13 A A A C G S14 C C G T A S15 A A A ? G S16 C C G C G ? indicates no PCR amplification, and the italic small letter indicates the allele with amplification of much lower efficiency. Table 5. Genotypes of genomic DNA, eight sperm aliquots and second-round MDA product of S16 at five microsatellite (STR) loci Samples Genomic DNA S04 S05 S06 S08 S13 S14 S15 S16 S16MDA2 D11S902 D11S937 D11S987 155/161 161 161 155 155 161 155 161 ? ? 156/168 156 168 156/168 168 168 156 168 156 156 94/116 94 ? 94 116 ? 94 116 94 94 D7S486 224/236 224 236 236 ? 236 ? 224 236 236 D10S547 244/254 244 254 254 254 254 244/254 254 244 244 ? indicates no amplification and the italic number in this table indicates the allele with amplification of much lower efficiency. DISCUSSION Four methods for WGA: PEP (23), degenerate oligonucleotide primed-PCR (DOP-PCR) (29), ligation-mediated PCR (LM- PCR) (30) and MDA (24), were developed and used for studies on single or mutiple cells. (16,25,27,30–32). Only a single primer was used in LM-PCR in contrast to the DOP and PEP, in which millions of different primers were included, LM-PCR can thus amplify whole genomic sequences more evenly and produce less bias than DOP and PEP (33), although it requires more steps, including restriction endonuclease digestion and ligation of digested DNA with primers. PEP was ﬁrst reported by Zhang et al. (23) to amplify single sperm DNA and then utilized by other researchers for single sperm analysis (15,16). Recently developed MDA was demon- strated to have a high ampliﬁcation potential and great loci representation with less than threefold bias (24). It has been successfully applied for WGA from laser capture- microdissected fresh tissue as well as single leukocytes and blastomere cells (25,26,34), although it was reported to fail in amplifying highly degraded samples, such as formalin-ﬁxed, parafﬁn-embedded tissues (35,36). The MDA method was used here to amplify whole genome DNA from single sperm cells and achieved high efﬁciency. In this study, when 1 ml of 1/50C0 MDA products were used as PCR templates, 76% reactions succeeded, and when 1 ml of 1/5C0 was used, the rate increased to 93%. This means that in a 50 ml reaction, 76 or 93% of loci could be ampliﬁed at least 2500- or 250-fold, respectively, from single sperm DNA, in contrast to only 78% of loci ampliﬁed 30-fold by the PEP method (23). Second-round MDA product analysis showed that a further 236-fold ampliﬁcation could be obtained. Con- sidering MDA is isothermally performed and can be done in an incubator, the MDA reaction volume may be extended to 100 ml or more. Pre-ampliﬁcation of the single sperm genome can therefore provide sufﬁcient DNA for thousands of geno- typing reactions. Our study used real-time PCR to quantitate the copy number for nine unique genes in 10 MDA products. Variable ampli- ﬁcation was observed. This variation seemed to result from random template selection, not from gene difference. For eight genes, copy number varied from zero to several thousand or more in 1 ml of 1/50C0 MDA products from different aliquots. Only one gene (PMS2) showed similar ampliﬁcation efﬁcien- cies across all eight samples. Preferred ampliﬁcation and allele dropout were observed to be quite common in MDA products from single leukocyte cells (data not shown). These were consistent with observations reported from other studies on single or a small number of cells (25,26). Many researchers have used WGA products from a limited number of cells (<5) for CGH or LOH analysis (26,30,31). Based on data from this study, we suggest much care should be taken in explaining these CGH or LOH data. Our sequencing data showed that MDA products were appropriate for sequencing projects. Here, a total of 27.4 kb nucleotides of MDA products from single sperm cells were