e91 Nucleic Acids Research, 2005, Vol. 33, No. 10 PAGE 4 OF 9 Figure 1. PCR amplification of the three fragments of the TOP1, P53 and CYP1A2 genes in MDA products of 16 sperm aliquots treated using two different lysis methods. The fragments are 1080 bp in length for TOP1, 643 bp for P53 and 550 bp for CYP1A2. Eight aliquots from the dilution of 3 cells/3 ml were lysed on ice and the other eight were lysed at 65 C. Figure 2. PCR amplification of the three fragments of the TOP1, P53 and CYP1A2 genes in MDA products of S01–S16 samples. The fragments are 1080 bp in length for TOP1, 643 bp for P53 and 550 bp for CYP1A2. S01–S16 indicate MDA products from 16 sperm aliquots. S16MDA2 is the second-round MDA product using 1 ml of 1/10C0 S16 MDA product as resource DNA. BLANK here is the MDA product of the negative control, in which 3 ml PBS buffer was added instead of sperm aliquot. alkaline lysis. In contrast, of the eight aliquots lysed on ice, only two have all three genes ampliﬁed, one aliquot has two and three have just one ampliﬁed. Moreover, many fragments showed much fainter bands than those ampliﬁed from MDA products of aliquots lysed at 65 C, indicating a lower efﬁciency of MDA in those samples lysed on ice. We thus selected incubation at 65 C for alkaline lysis of 16 ali- quots (S01–S16) from the dilution of 0.8 cells/3 ml, which were selected for detailed analysis of efﬁciency of genome ampli- ﬁcation of single sperm cell by MDA. All of the three genes tested (TOP1, P53 and CYP1A2) were successfully ampliﬁed in 7 of 16 sperm aliquots at the 0.8 cells/ 3 ml dilution (S01, S04, S05, S06, S14, S15 and S16). For S08 and S13, only TOP1 and CYP1A2 genes were ampliﬁed (Figure 2). For the others, no ampliﬁcation was observed. A total of 9 out of 16, or 56%, sperm aliquots were therefore selected for further studies. This observation was consistent with the expectation of cumulative Poisson distribution (55%) with a mean of 0.8 for aliquots with at least one sperm, so Figure 3. PCR amplification of 12 fragments using 1 ml of 1/5C0 or 1/50C0 MDA products as templates. The amplification result is indicated with a black square for successful amplification in 1/50C0, a gray square for successful amplification in 1/5C0 and a framed F for no amplification in both. most aliquots containing sperm cell(s) should be ampliﬁed, although we could not estimate the success rate. Two of them (S06 and S14) may have contained more than one sperm based on further sequencing and STR genotyping below. Coverage The coverage of the ampliﬁcation can be estimated based on the success rate of ampliﬁcation of the 12 fragments (see Materials and Methods) tested. Most of the 12 fragments were successfully ampliﬁed in all nine sperm aliquots (Figure 3). For those seven aliquots containing single sperm cells (S01, S04, S05, S08, S13, S15 and S16), the success rate ranges from 58 to 92% for 1/50C0 with an average of 76%. If 1/5C0 MDA products were used, the coverage increased to 93%. About 76% of the genes can therefore be ampliﬁed at least 2500-fold or 93% of the genes can be ampliﬁed at least 250-fold in single sperm cell suspensions. The second-round MDA product, S16MDA2, from sample S16, had almost the same ampliﬁcation pattern. Yield MDA yields for the nine aliquots containing sperm and the S16MDA2 product were 13–35 mg (Table 2), and the yields for other aliquots without sperm were 15–35 mg. It was clear that background ampliﬁcation in MDA was quite high, so total DNA yield did not provide much information on MDA efﬁ- ciency, and thus a detailed quantitative analysis became neces- sary. Here, we quantitated nine genes in MDA products of nine sperm aliquots and the S16MDA2 (Table 2). Although the speciﬁc fragment was ampliﬁed in S16 and S16MDA2 for P53, S15 for CYP1A1, S13 for C6orf195 and S13 for ADCYAP1, another unspeciﬁc fragment was also observed in each of these reactions. The unspeciﬁc ampliﬁcation makes it impossible to determine the accurate copy number. We, therefore, considered these reactions as failed and estim- ated the gene copy number based on PCR results in Figure 3. The PIK3CA gene, which was not ampliﬁed from 1 ml of 1/50C0 S13 or S15 MDA product in the coverage test, was estimated to have two or four copies. This inconsistency occurred because the PCR condition for amplifying the PIK3CA fragment in the coverage test was possibly not