e91 Nucleic Acids Research, 2005, Vol. 33, No. 10 the whole genome level in 1992 (23) and ﬁrst applied for localization of recombination sites in 1994 (15). Recently, Cullen et al. (16) used PEP to pre-amplify 20 031 single sperm cells, followed by genotyping using 48 short tandem repeat (STR) markers in a 3.3 Mb interval encompassing the major histocompatibility complex (MHC). Unfortunately, their study did not achieve high enough density to allow com- parison with the LD map, owing to insufﬁcient PEP products and low marker density. Recently, multiple displacement ampliﬁcation (MDA), using F29 DNA polymerase and random exonuclease- resistant hexamer primers, has been demonstrated to be very efﬁcient for balanced ampliﬁcation and generation of long DNA products (>10 kb) from a small amount of DNA, or directly from whole blood (24). The high processivity and ﬁdelity of F29 DNA polymerase provides an advantage to MDA in terms of yield, accuracy and coverage over the other PCR-based WGA methods (24,25). Ampliﬁcation of single lymphocytes or blastomeres using MDA has been successfully carried out (26,27). Up to 92% coverage was estimated from 100 PCRs by detecting 20 different loci in ﬁve single lympho- cytes, although at heterozygous loci, a high rate of allele dropout (31%) was observed (26). In this paper, we demonstrate that MDA can be utilized to produce high-coverage and high-yield WGA products from single sperm cells, and the resulting MDA products can be used for a variety of genetic applications, including sequen- cing and microsatellite marker (STR) and single nucleotide polymorphism (SNP) analysis. It was estimated that 76% of the genomic sequence can be ampliﬁed at least 2500-fold, or 93% at least 250-fold. We also showed that the second-round MDA can further give an averaged 236-fold ampliﬁcation. MDA can therefore be quite useful for amplifying single sperm DNA for a large amount of genotyping reactions and this may open a new avenue for single sperm analysis. MATERIALS AND METHODS Sperm sample and genomic DNA preparation Fertile men considering vasectomy were approached for consent to enter the study. Once informed consent was obtained, a blood sample and semen sample (by masturbation) were collected. The blood sample (10 ml) was collected into tubes with acid citrate dextrose (ACD) and centrifuged at 3300 g at room temperature for 10 min. The intermediate layer where white blood cells were concentrated was collected and resuspended in phosphate-buffered saline (PBS) for further processing for DNA analysis. Genomic DNA was extracted from white blood cells using the standard phenol– chloroform method. DNA concentration was determined using a Hoefer DyNA Quant 200 Fluorometer. Sperm lysis Sperm cells were counted with a hemacytometer, diluted to a concentration of either 0.8 or 3 cells/3 ml with PBS and 16 aliquots were prepared of each dilution. Three microliter of diluted sperm cells were dispensed into 200 ml PCR tubes and frozen at 80 C overnight. An aliquot of 3.5 ml of freshly prepared lysis solution (0.1 M DTT, 0.4 M KOH and 10 mM PAGE 2 OF 9 EDTA) was then added, mixed well by gentle vortex and incubated for 10 min on ice for eight aliquots of the dilution of 3 cells/3 ml, or at 65 C for the other aliquots. Lysis was stopped by adding 3.5 ml of neutralizing buffer (buffer B in REPLI-g kit, Qiagen Inc.). The dilution of 3 cells/3 ml was picked to test whether 65 C incubation could lyse sperm cells better or not, and the dilution of 0.8 cells/3 ml was used to obtain aliquots containing single sperm cells. Aliquots named after S01, S02. . .S16 below were prepared from the dilution of 0.8 cells/3 ml Multiple displacement amplification WGA was achieved using REPLI-g kit according to the manufacturer’s manual (Qiagen Inc.). All samples were pre- ampliﬁed by MDA. A PBS blank was included as a negative control. A reaction in a total volume of 50 ml was performed at 30 C overnight and then terminated at 65 C for 10 min. Ampliﬁed DNA products were then stored at 20 C. Dilu- tions of 5- or 50-fold (referred as 1/5C0 and 1/50C0, respect- ively, below) were used for further sequencing, the coverage test and microsatellite and SNP genotyping analysis. One microliter of a 10-fold diluted S16 MDA product was used as template for the second-round MDA. PCR and sequencing analysis In order to determine the aliquots that were successfully pre- ampliﬁed by MDA, three genes—TOP1, P53 and CYP1A2— were selected for PCR testing using 1 ml of 1/5C0 MDA prod- uct. Primers used are listed in set A of Table 1. A 20 ml mixture was prepared for each reaction and included 1· HotStarTaq buffer, 2.5 mM Mg2+, 0.2 mM dNTP, 0.3 mM of each primer, 1 U HotStarTaq polymerase (Qiagen Inc.) and 1 ml template DNA. 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 1 min; 72 C for 2 min. Ampliﬁed fragments representative of the three genes (TOP1, P53 and CYP1A2) were 1080, 643 and 550 bp in length, respectively. PCR products were checked on 1.5% agarose gels. For the aliquots of the 0.8 cells/3 ml dilution, those MDA products in which at least one of the three genes got ampliﬁed were selected for further analyses. A total of 12 genes, including TOP1, P53, CYP1A1, PIK3CA, C6orf195, DKKL1, SHH, ADCYAP1, MSH2, PTEN, PMS2 and CAT, were examined to estimate ampliﬁ- cation coverage. Almost all genes are located on different chromosomes, except for SHH and PMS2, which are both on chromosome 7 (PMS2 on the p arm and SHH on the q arm). The standard PCR method described above was used to amply 12 sequences of length 162–351 bp, one for each gene, from 1 ml of 1/50C0 MDA product. Primers used to amplify these fragments are listed in set B of Table 1. Five microliter PCR products were then loaded on 1.5% agarose gels and checked for the presence of target fragments. For those sam- ples in which some primers did not work, the 1/5C0 MDA products were submitted to PCR again with the failed primers. To assess the ﬁdelity of WGA from single cells, we used another four pairs of primers (set C in Table 1) to amplify four DNA fragments (two in TOP1 and two in CYP1A1) with a total length of 3.3 kb, and these fragments were then sequenced using ABI Big Dye Terminator V3 kit and ABI3100 capillary sequencer (AppliedBiosystems, Foster