We have developed an in vitro method for amplifying a large fraction of the DNA sequences present in a single haploid cell by repeated primer extensions using a mixture of 15-base random oligonucleotides. We studied 12 genetic loci and estimate that the probability of amplifying any sequence in the genome to a minimum of 30 copies is not less than 0.78 (95% confidence). Whole genome amplification beginning with a single cell, or other samples with very small amounts of DNA, has significant implications for multipoint mapping by sperm or oocyte typing and possibly for genetic disease diagnosis, forensics, and the analysis of ancient DNA samples.

The use of the polymerase chain reaction for analysing DNA sequences in individual diploid cells and human sperm shows that two genetic loci can be co-amplified from a single sperm, which may allow the analysis of previously inaccessible genetic phenomena.

The frequency of recombination between the Gy-globin (HBG2) and parathyroid hormone (PTH) loci on the short arm of human chromosome 11 was estimated by typing >700 single-sperm samples from two males. The sperm-typing technique employed involves the polymerase chain reaction and allele-specific oligonucleotide hybridization. Our maximum likelihood recombination fraction estimate of 0.16 (95% confidence interval, 0.13-0.19) falls well within previous estimates based on family studies. With current technology and a sample size of 1000 sperm, recombination fractions down to =0.009 can be estimated with statistical reliability; with a sample size of 5000 sperm, this value drops to about 0.004. Reasonable technological improvements could result in the detection of recombination frequencies <0.001.The determination of genetic linkage between polymorphic markers in humans has traditionally depended upon pedigree analysis. Such studies have a number of inherent difficulties when compared to linkage analysis in experimental organisms. Humans of the appropriate genotypes cannot be crossed at will. In those crosses that are available, linkage phase is often unknown; this uncertainty blurs the distinction between recombinants and nonrecombinants. Finally, the difficulty in obtaining large numbers of offspring from families informative for a particular set of genetic markers effectively limits statistically reliable recombination frequency estimates to 0.01 or 0.02 (i.e., 1 or 2 centimorgans). A radically different approach to measuring recombination frequency has been suggested (1) that has a number of advantages when compared to traditional family studies. This approach has, as its basis, the direct molecular analysis of DNA sequences in single meiotic products. The method uses the polymerase chain reaction (PCR) (2-4) to amplify polymorphic DNA sequences at two or more genetic loci in a single sperm. Analysis of single human sperm by this typing procedure has already demonstrated Mendelian segregation of alleles at each of two loci and independent assortment of genes on nonhomologous chromosomes (1). With sperm typing a direct count of recombinant and nonrecombinant meiotic products can be made and used to estimate recombination frequency. Since a single human semen sample contains >300 million sperm, this approach is not limited by sample-size considerations and is capable of high-resolution genetic mapping.Before embarking on studies to measure very small recombination fractions using the sperm-typing approach, we thought it prudent to estimate the frequency of recombination between two loci whose genetic distance from one another had already been determined by pedigree analysis. We have, therefore, estimated by sperm typing the recombination fraction between the parathyroid hormone gene locus (PTH) and the Gy-globin gene locus (HBG2) on the short arm of human chromosome 11. In these experiments, we examined 708 sperm samples from two males. We have also developed a mathematical approach for analyzing sperm-typi...

A high-throughput genotyping system for scoring single nucleotide polymorphisms (SNPs) has been developed. With this system, >1000 SNPs can be analyzed in a single assay, with a sensitivity that allows the use of single haploid cells as starting material. In the multiplex polymorphic sequence amplification step, instead of attaching universal sequences to the amplicons, primers that are unlikely to have nonspecific and productive interactions are used. Genotypes of SNPs are then determined by using the widely accessible microarray technology and the simple single-base extension assay. Three SNP panels, each consisting of >1000 SNPs, were incorporated into this system.The system was used to analyze 24 human genomic DNA samples. With 5 ng of human genomic DNA, the average detection rate was 98.22% when single probes were used, and 96.71% could be detected by dual probes in different directions. When single sperm cells were used, 91.88% of the SNPs were detectable, which is comparable to the level that was reached when very few genetic markers were used. By using a dual-probe assay, the average genotyping accuracy was 99.96% for 5 ng of human genomic DNA and 99.95% for single sperm. This system may be used to significantly facilitate large-scale genetic analysis even if the amount of DNA template is very limited or even highly degraded as that obtained from paraffin-embedded cancer specimens, and to make many unpractical research projects highly realistic and affordable.

We have developed a procedure that allows the detection of polymerase chain reaction (PCR) products derived from a single target DNA molecule in a human sperm without using radioactive probes. With this method, three genetic loci present in a single sperm can be amplified simultaneously. The amplification procedure is specific as well as efficient and permits detection of the PCR product by ethidium bromide staining after polyacrylamide gel electrophoresis. When allele-specific PCR primers that differ in length are used, the size of the PCR products of different alleles also vary in length, allowing the allelic state at each locus to be determined electrophoretically. Studies on individual sperm by using this procedure should facilitate the measurement of genetic recombination in humans over small physical distances. The ability to directly analyze the allelic state of PCR products from one cell rapidly and simply will also be useful for the prenatal diagnosis of genetic disease, especially in the analysis of single blastomeres taken from in vitro fertilized eggs prior to implantation.

To facilitate large-scale genotype analysis, an efficient PCR-based multiplex approach has been developed. For simultaneously amplifying the target sequences at a large number of genetic loci, locus-specific primers containing 5' universal tails are used. Attaching the universal tails to the target sequences in the initial PCR steps allows replacement of all specific primers with a pair of primers identical to the universal tails and converts the multiplex amplification into "uniplex." Simultaneous amplification of 26 genetic loci with this approach is described. The multiplex amplification can be coupled with genotype determination. By incorporating a single-base mismatch between a primer and the template into the target sequences, a polymorphic site can be converted into a desirable restriction fragment length polymorphism when it is necessary. In this way, the allelic PCR products for the polymorphic loci can be discriminated by gel electrophoresis after restriction enzyme digestion. In this study, 32 loci were typed in such a multiplex way.As the human genome project progresses rapidly, expanding experimental scale in genotyping has become an increasing demand for human genetic mapping, genetic disease diagnosis, mutation screening, and genome-scale search for diseasecausing genes. For example, according to a recent report (1), nearly 6000 genetic markers have been placed on the comprehensive map, and how to fully utilize these markers on the map and the map information has become a major issue. With currently available experimental approaches, typing a single sample at all marker loci on the map would be very costly and laborious. Although automation can greatly reduce the amount of hands-on work, it cannot reduce the cost for reagents and supplies. Therefore, developing multiplex approaches for large-scale genotype analysis is a necessary step and the only efficient way to reduce the cost for fully utilizing the markers and the map information.Among genetic markers, PCR-based markers can be used easily and cost effectively. For these markers, genotype determination is carried out in two steps: (i) amplifying the polymorphic DNA regions by PCR and (ii) determining the sample genotype by analyzing the PCR products. Multiplexing the second step has been relatively easy by mixing multiple PCR products before electrophoresis (2, 3), dot-blotting multiple samples to a membrane before probing (4, 5), or multiple pipetting with a robotic device (6). By sequentially loading sample sets to a gel during electrophoresis after restriction enzyme digestion, the allelic products of a large number of sample sets can be sequentially resolved (7). However, the scale of multiplex amplification has been limited mainly due to the serious primer-primer interaction when multiple sets of primers are present in the same reaction mixture. With carefully optimized experimental conditions, only -10 loci could be amplified simultaneously (for review, see ref. 8).The publication costs of this article were defrayed in part by pa...

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BackgroundSegmental duplication and deletion were implicated for a region containing the human immunoglobulin heavy chain variable (IGHV) gene segments, 1.9III/hv3005 (possible allelic variants of IGHV3-30) and hv3019b9 (a possible allelic variant of IGHV3-33). However, very little is known about the ranges of the duplication and the polymorphic region. This is mainly because of the difficulty associated with distinguishing between allelic and paralogous sequences in the IGHV region containing extensive repetitive sequences. Inability to separate the two parental haploid genomes in the subjects is another serious barrier. To address these issues, unique DNA sequence tags evenly distributed within and flanking the duplicated region implicated by the previous studies were selected. The selected tags in single sperm from six unrelated healthy donors were amplified by multiplex PCR followed by microarray detection. In this way, individual haplotypes of different parental origins in the sperm donors could be analyzed separately and precisely. The identified polymorphic region was further analyzed at the nucleotide sequence level using sequences from the three human genomic sequence assemblies in the database.ResultsA large polymorphic region was identified using the selected sequence tags. Four of the 12 haplotypes were shown to contain consecutively undetectable tags spanning in a variable range. Detailed analysis of sequences from the genomic sequence assemblies revealed two large duplicate sequence blocks of 24,696 bp and 24,387 bp, respectively, and an incomplete copy of 961 bp in this region. It contains up to 13 IGHV gene segments depending on haplotypes. A polymorphic region was found to be located within the duplicated blocks. The variants of this polymorphism unusually diverged at the nucleotide sequence level and in IGHV gene segment number, composition and organization, indicating a limited selection pressure in general. However, the divergence level within the gene segments is significantly different from that in the intergenic regions indicating that these regions may have been subject to different selection pressures and that the IGHV gene segments in this region are functionally important.ConclusionsNon-reciprocal genetic rearrangements associated with large duplicate sequence blocks could substantially contribute to the IGHV region diversity. Since the resulting polymorphisms may affect the number, composition and organization of the gene segments in this region, it may have significant impact on the function of the IGHV gene segment repertoire, antibody diversity, and therefore, the immune system. Because one of the gene segments, 3-30 (1.9III), is associated with autoimmune diseases, it could be of diagnostic significance to learn about the variants in the haplotypes by using the multiplex haplotype analysis system used in the present study with DNA sequence tags specific for the variants of all gene segments in this region.