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10.1.1 Single sperm cell typing

In 1988, Arnheim and his colleagues (Li et al., 1988) reported the extraordinary finding that unique DNA sequences could be amplified from isolated sperm cells. Amplification from the single DNA target locus that is present in haploid sperm cells represents the theoretical limit in sensitivity that is possible with PCR. The implications of this experimental breakthrough were enormous, especially in the field of human genetics. For the first time, it became possible to consider the analysis of unlimited numbers of individual meiotic events derived from a single individual.

Linkage information could be derived by first identifying a male volunteer who was heterozygous at all loci of interest in a manner that could be distinguished with the use of PCR. The DNA within individual sperm cells donated by the volunteer would be subjected to co-amplification with primer pairs that define each of the loci. Allele determination at each locus in each cell could, in theory, be accomplished by any of the various PCR techniques described in Section 8.3, including hybridization to allele specific oligonucleotides, restriction enzyme digestion, or SSCP. In contrast to typical genetic studies in humans, the typing of large numbers of single sperm cells from a single volunteer provides genetic information which is simple to interpret. Linkage distances and gene order can be derived directly by counting the number of cells with each allelic combination (Goradia et al., 1991), in a manner equivalent to that described in (Section 9.4) for the analysis of backcross data in the form of haplotypes. Since the number of sperm cells is essentially unlimited, the resolution of the map obtained is a function simply of the time and effort that the investigator wishes to expend in the typing of additional cells. Furthermore, with the use of a protocol for universal PCR amplification, Arnheim and his colleagues have been able to co-amplify the majority of sequences present in each sperm genome (Zhang et al., 1992). Thus, in theory, it should be possible to co-type many different loci within a single panel of sperm samples.

Although single sperm typing provides a significant new tool for mapping in humans and other large animals that also do not provide a sufficient number of offspring for typing, its importance to mouse genetics would appear to be more limited since numerous, well defined backcross mapping panels are available for analysis. Furthermore, at the time of this writing, the typing of single cells is still so technically-demanding that it has not been used in a general way even by the broader community of human geneticists.

10.1.2 Mitotic linkage maps

Classical linkage analysis is based on meiotic recombination events that occur in sperm and egg precursor cells. The products of these events are observed and counted in the offspring of heterozygous animals (or in sperm cells as described in the previous section). Meiotic recombination is a very frequent event — it occurs 30 times, on average, in each individual germ cell line — and it appears to have been selected by evolution to play two very different roles in the biology of higher eukaryotes. First, the physical event itself appears to be required to tether the homologs of each chromosome to each other so that they line up and segregate into opposite daughter cells during the first meiotic division. Second, the production of offspring with non-parental combinations of alleles provides a non-mutational means for the generation of diversity at the genotypic level, and this generation of diversity appears to be generally beneficial to the population as a whole.

Recombination has also been observed at the mitotic level in somatic cells (Rajan et al., 1983; Henson et al., 1991). In comparison to meiotic recombination, mitotic events are exceedingly rare, and they do not appear to have any biological function. It seems most likely that mitotic recombination events are simply accidents that happen in response to spontaneous nicks in the DNA molecule that allow migrating single strands to invade opposite homologs. Usually, mitotically recombined cells will go entirely unnoticed among the millions of nearby cells having germ line haplotypes. However, in individuals that are born heterozygous for null alleles at "tumor suppresser genes" such as retinoblastoma (RB), mitotic recombination can lead to the production of rare homozygous mutant cells that are released from growth control in the absence of the wild-type allele; uncontrolled division of these cells leads to tumor formation. It now appears that a large class of human tumors are caused by this homozygosing at a variety of tumor suppresser genes (Marshall, 1991; Weinberg, 1991).

If rare cells that undergo mitotic recombination could be identified and recovered in clonal form from a tissue culture line, a means for generating a linkage map that was not dependent on the breeding of animals would be possible. Such a "mitotic linkage map" has been obtained for the proximal half of chromosome 17 (Henson et al., 1991). The generation of this particular map was dependent upon the ability to immuno select cells that had undergone allele loss at an H-2 complex gene. Selected cells were isolated and expanded into cultures that could be analyzed for various DNA markers that were heterozygous in the parent. All loci that map proximal to the clone-specific breakpoint will remain heterozygous; all that map distal will have become homozygous. Through the analysis of a large number of individual clonal lines, it becomes possible to construct a linkage map with gene order and an estimate of relative distances between loci. The mitotic linkage map constructed for the proximal half of chromosome 17 corresponds well with the meiotic linkage map (Henson et al., 1991). As expected, the gene order is identical, and there is some minor variation in the relative intergenic distances.

The construction of a mitotic linkage map in one chromosomal region was important for providing biological information concerning the relationship between the distribution of mitotic and meiotic crossover sites, and future experiments of this type may also provide clues to the mechanisms responsible for homologous recombination in somatic cells. However, the mitotic map did not provide any new information specific to chromosome 17, and this approach is not likely to play an important role in future gene mapping experiments for two reasons. First, with current technology, mitotic maps can only be constructed along chromosomal regions that are marked with genes encoding polymorphic cell surface antigens that are expressed codominantly in cultured cells, and are readily distinguishable from each other with specific monoclonal antibodies. 103 The second reason is that the construction of traditional meiotic linkage maps at the same resolution is likely to be much faster and easier.

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