PAGE 3 OF 9 Table 1. Primers for PCR in this study Set A Set B Set C Fragments TOP1 P53 CYP1A2 TOP1 P53 CYP1A1 PIK3CA C6orf195 DKKL1 SHH ADCYAP1 MSH2 PTEN PMS2 CAT TOP1 (1) TOP1 (2) CYP1A1 (1) CYP1A1 (2) Nucleic Acids Research, 2005, Vol. 33, No. 10 e91 Forward primers 50-CCCCTAGATTGATGCCAAGA-30 50-CTCATCTTGGGCCTGTGTTA-30 50-CAGAATGCCCTCAACACCTT-30 50-TGACTCATCTCTTATGGTTGCAG-30 50-GCCTCTTGCTTCTCTTTTCC-30 50-ATTTCCAGCTGCTGTCACAT-30 50-TTTGCTCCAAACTGACCAAAC-30 50-TGATAGGCATTTCTTTTGAAACTGG-30 50-CATCTGCAAAGTGGGCTGAATAGG-30 50-TCTCGGAACTCAATGCCCTGTC-30 50-CAAGTGCTGTTCAACTCAGGGA-30 50-TCCATTGGTGTTGTGGGTGT-30 50-TGAAGACCATAACCCACCACAG-30 50-CTGGATGCTGGTGCCACTAA-30 50-TACCAGCTCCAGTGGTCAGG-30 50-GCATGAATCTCAGCTCTTTCCA-30 50-GATGACTTGGGCTCTCCCTTT-30 50-CTGCAGCCAGATCAGTGTCTA-30 50-TTTTGCGTATTTATGTTGCAGA-30 Reverse primers 50-ACTTTAGCCCGGACTAGCAA-30 50-ACCGCTTCTTGTCCTGCTT-30 50-TCACACAGCTGGTCTGAGGT-30 50-CAACCCACTGCTGAATGATG-30 50-ACCGCTTCTTGTCCTGCTT-30 50-CTGGTCTCCAGCTGCCTTTA-30 50-ACTCCAAAGCCTCTTGCTCA-30 50-ACAGGGAAGCAAAGCACTTC-30 50-CCGTGGCAGCTTAATGATCC-30 50-GGTCCAGGAAAGTGAGGAAG-30 50-GGCGATGCTAGTAGTCTGGACC-30 50-TCAGTTTCCCCATGTCTCCA-30 50-CTAGGGCCTCTTGTGCCTTT-30 50-GCCTGGCACACCGTAAGAA-30 50-CCCATCCTGTCAGATTTTAGTACTTT-30 50-GATGGGTTAGGCCCTCTTAAA-30 50-GGGGAAACAGCATTGATCCTA-30 50-GACACAGTGATTGGCAGGTC-30 50-AGGCTCTGCTTTGATTGAGG-30 City, CA). PCRs were prepared and performed as described above. These four fragments were selected because they include ﬁve SNPs: rs6129731, rs2235362, rs6102287, rs1048943 and rs17861120, which are heterozygous in the studied sample, so the sequencing data can also provide hap- lotype information and help check whether the MDA products does come from single sperm cells or not. Quantification of gene copy number We quantiﬁed the copy number for nine genes with the abso- lute quantiﬁcation program of SDS2.0 software using an ABI7900 machine. These nine genes were same as those used in MDA coverage analysis, except for three genes, DKKL1, SHH and MSH2, which were excluded owing to bad standard curves. Primers for quantiﬁcation were same as those used for coverage analysis. Human genomic DNA from Promega (Catalog no. G1521) was serially diluted as input DNA for the construction of standard curves. Each reac- tion was duplicated three times. A 20 ml mixture was prepared for each reaction and included 0.05· SYBR (Molecular Probes, Eugene, OR), 1· HotStarTaq buffer, 2.5 mM Mg2+, 0.2 mM dNTP, 0.3 mM of each primer, 1 U HotStarTaq poly- merase (Qiagen Inc.) and 1 ml of 1/50C0 MDA products. The cycling program was 95 C for 15 min; 40 cycles of 94 C for 15 s, 56 C for 30 s, 72 C for 30 s. We determined the total number of gene copies ampliﬁed and their standard devia- tions in a 50 ml reaction by multiplying those in 1 ml of 1/50C0 MDA products by 2500. For those samples in which real-time PCR did not work, 0, 250 and 2500 were assigned to those samples with no ampliﬁcation or ampliﬁed in 1/5C0 or 1/50C0 MDA products in the coverage test, respectively. STR and SNP genotyping In order to test whether the WGA products from single cells were useful for STR and SNP genotyping analysis, ﬁve STR markers (D11S902, D11S937, D11S987, D7S486 and D10S547) were typed using the ABI 3100 sequencer and seven SNPs (rs4575595, rs11868324, rs4267385, rs5369, rs1800541, rs1535721 and rs1323690) were examined with Taqman Assay-on-demand kits (AppliedBiosystems). The S01 sample was not included owing to accidental loss. All these markers were heterozygous in the sampled individual. One microliter of 1/5C0 MDA products were added as tem- plates in the reactions. Five microsatellite sites were separately ampliﬁed in a 6 ml reaction containing 1· HotStarTaq buffer, 2.5 mM Mg2+, 0.2 mM dNTP, 0.3 U HotStarTaq polymerase (Qiagen Inc.), 0.06 ml of ﬂuorescence-labeled primer mixture from ABI PRISM Linkage Mapping Sets V2.5 and 1 ml of template DNA. The cycling program was 95 C for 15 min; 10 cycles of 94 C for 15 s, 55 C for 15 s, 72 C for 30 s; 28 cycles of 89 C for 15 s, 55 C for 15 s, 72 C for 30 s; 72 C for 1 min. The PCR products for D11S987, D11S937 and D7S486 or for D10S547 and D11S902 were mixed for subsequent gel load- ing. Data for STR markers were collected with the 3100 Data Collection V1.1 software and analyzed with GeneMapper V3.0. PCR for Taqman SNP typing was performed in a 5 ml reaction containing 2.5 ml 2· Taqman universal PCR master mixture, 0.125 ml 40· assay mixture and 1 ml template DNA. The cycling program was 95 C for 10 min; 45 cycles of 92 C for 15 s, 60 C for 1 min. Genotypes were determined by allelic discrimination program in SDS 2.1 software. RESULTS Successful ampliﬁcation of whole genomic DNA in cells by MDA depends on efﬁcient release of DNA from chromo- somes. DNA molecules are packed very densely in the sper- matozoon nucleus and a much tougher method is needed to isolate DNA from sperm cells than from other tissue cells (28). The manual for the REPLI-g kit suggests incubation on ice during alkaline lysis of blood or tissue culture cells. Here, we showed that incubation at 65 C could provide much better genome ampliﬁcation from minute sperm cells than incubation on ice (Figure 1). Eight aliquots from the dilution of 3 cells/3 ml were subject for alkaline lysis on ice and another eight aliquots were lysed at 65 C. All three genes tested (TOP1, P53 and CYP1A2) were shown to undergo ampliﬁcation successfully in seven out of eight aliquots if incubated at 65 C during