PAGE 5 OF 9 7 +5E +5E +5E +5E seo tsc + E th ud .20 .01 .20 .02 .03 DA .traeoF rpoDA 2 – – – – – 16S *0 2500* +.170E 25*0 +.105E +.250E +.529E +.518E 0* 30 41 15 itrev CM0 M a 0 +7E +5E +5E +5E +5E t en /15 t c rse dn u .10 .05 .20 .02 .01 d o rep a0 r p – – – – – DA /15C M 16S 50*2 2500* +.374E 25*0 +.245E +.651E +.558E +.545E 0* 14 295 105 ml anuDm itianon iecanh50 –+.053E –+.107E –+.104E –+.405E –+.015E –+.026E –+.075E –+.063E ..frohRR lliifacpm tv 15S +.413E +.217E 520*0 +.104E +.554E +.254E +.6617E +.957E +.531E 53 98 35 andgADN ,fssscecunu e a r e i t tan +7E +6E +3E +6E +5E +3E +5E +6E rrseeepA –+.027E –+.016E –+.253E –+.056E –+.115E –+.073E –+.025E –+.196E rreepbn iiltifcao DN 14S .26 .12 .71 *0 .32 .70 .44 .917 .37 26 158 56 num apm an py no um +4E +3E +6E +5E +6E eco ith t)eahhdnT –.05 –.10 –.03 –.20 –.04 rfrseaavgo rcaeccndow CA 13S 50*2 250* +.1534E +.503E 520*0 2500* +.366E +.2514E +.268E 92 30 11 /ign ian ,and2SPNM +–+.3503E +–+.6026EE +–+.6606EE +–+.4174EE +–+.6066EE +–+.6406EE +–+.4074EE ....traCNAm iltsseaedngy ,1PPETCAY 08S +..81402EE +.6*010E +..42104E +..72902E +..66101E +.50*03E +..64802E +..51606E +..62304E 32 40 14 tiiiiffscecnon rrsaeeeap00w ,,fr6o15911PCADA 06S ++––..616805EE ++––..412650EE ++––..6335407EE ++––..513706EE ++––..313603EE ++––..3817536EE ++––..332645EE ++––..696501EE +–+–..350614EE 26 774 276 tittrrrsaeeeeubnhnwm ,,tir)ssaae2500knd25 ,53CY 05S +.3206E +.614E +.396E +.415E +.893E +.883E +.563E +.216E +.993E 29 114 41 taand itdhw P ,1P +7E +5E +7E +5E +5E +6E +4E +6E +6E rom leeb (TO .01 .20 .01 .04 .01 .02 .02 .01 .01 –fSD lr(aok seegn +–7E +–5E +–7E +–5E +–5E +–6E +–4E +–6E +–6E ean tow inn 04S .21 .63 .39 .38 .31 .36 .323 .14 .16 20 379 135 iendm idndR e rfreunyobm +–.064E +–.550E +–.511E +–.6630E +–.406E +–.520E lisaypd iePCm a 01S +.226E +.425E +.555E 25*0 +.58E 250* +.3174E +.256E 2*05 13 80 29 rseaubopynwm itlr-secahhonw .iireendgu3F p o c l t oT 1 .le2abT 1PTO 53P 11PCYA I3PCKA fr16o95C PCADYA PETN 2PSM TCA mli)(edgY .../gnCAN ..)(RR% ltacoT lsaepm lisaydp Nucleic Acids Research, 2005, Vol. 33, No. 10 e91 optimized for several template molecules. Gene copy number in these MDA products varied greatly across samples and gene loci. In the 50 ml MDA products for seven single sperm cells (S01, S04, S05, S08, S13, S15 and S16), 51% of fragments could be ampliﬁed at least 250 000-fold. The human DNA representative rate (R.R. in Table 2) in MDA products was estimated as observed average copy number per nanogram DNA (A.C.N./ng in Table 2) divided by 280. Here, 280 is considered as the copy number of unique genes per nanogram human genomic DNA (Qiagen Genomic DNA Handbook). Some genes were over-ampliﬁed in some sperm samples, which caused the human DNA representative rate to be >100% in the ampliﬁed sperm samples, such as S04, S06 and S16. The representative rate for second-round MDA product was reduced from 105 to 15% for S16MDA2. Even though the representative rate for second-round MDA samples was dra- matically decreased, the second-round MDA can give an amp- liﬁcation of from 0- to 500-fold for eight genes, with an average of 236-fold (Table 3). Sequencing We sequenced four fragments (overall 3.3 kb) in all 10 MDA products (nine sperm aliquots and one second-round MDA product) and the leukocyte genomic DNA. Only one artiﬁcial mutation was observed, in sample S14 (Figure 4A). The error rate of MDA ampliﬁcation in a single or a few sperm cells was therefore estimated to be 3.65 · 10 5 in an overall 27.4 kb sequenced. All MDA products from sperm aliquots, except S06 showed single alleles at ﬁve SNP sites in the four fragments (Table 4). Rs1048943 and rs17861120 were observed to be heterozygous in S06, although the peak height of one allele was much lower that that of the other (Figure 4B, rs1048943). Together with the STR data (see below), S06 and S14 were deduced to contain more than one sperm, whereas the other seven aliquots were considered to have just one sperm each. Rs6129731, rs2235362 and rs6102287 are in the same Contig NT_011362.9. Rs1048943 and rs17861120 are in another Contig NT_010194.16. From Table 4, it is obvious that haplotypes for the ﬁrst three SNPs are AAA and CCG, and haplotypes for the latter two SNPs are CG and TA. Genotyping Five STRs were successfully typed with the ABI 3100 sequen- cer and GeneMapper software in 59 out of 65 reactions. The genotypes for the nine sperm aliquots and S16MDA2 are described in Table 5. S06 and S14 were shown to be hetero- zygous at D11S937 and D10S547, respectively (Figure 4C, D10S547). The allele 168 for D11S937 and 254 for D10S547 were preferentially ampliﬁed. S06 was also observed to be heterozygous at two SNP sites as described in Table 4. This indicated that the two aliquots contained at least two sperm cells. Seven SNPs located in three genes, ACE, EDN1 and ELF5, were successfully typed on the ABI7900 machine with Assay-on-demand kits in 84/91 reactions (Figure 4D, rs1535721). Genotypes for each sperm aliquot are described in Table 6. Haplotypes for ACE, EDN1 and ELF5 were easily determined to be GAT/AGC, GT/AG and AG/GA, respect- ively. S16MDA2 had exactly the same genotypes with S16 sample for all STRs and SNPs examined.