In situ Hybridization

Human-Rodent Somatic Cell Hybrids

Recombination Mapping of Meiotic Chromosomes

Fine Structure Mapping of Chromosomes

Map-based or Positional Cloning of Genes

Study Questions

Genomic Analysis WWW Links

Genetic Topics

Meiotic Recombination Mapping

As discussed earlier in the course, the linkage relationship between two or three genes or markers can be determined by anlayzing the number of recombination events that occur in a specific mating of individuals polymorphic for the genes in question. In general, the term for this procedure is called meitotic recombination mapping. The same scientific principles apply when developing a detailed map of any chromosome. The only difference is that the segregating population is not scored for just two or three genes but for a large number of genes.

To develop a species map from a given population, the parents used to develop the population must be polymorphic at many genes or loci. It is often difficult to get two parents that differ from each other by all of the important genes. The primary reason is that as you accumulate more and more mutants alleles in an individual, the fitness of the individual declines to the point that the individual becomes non-fertile. The advent of molecular markers has alleviated this problem. The advantage of molecular markers is that they are not lethal. Therefore all that is necessary to develop the map is to identify two parents that are polymorphic for many molecular markers. Molecular markers can be considered to be sign posts along the chromosome of the species. So mapping molecular marker loci will give you a molecular map of the species similar to a genetic map.

Several types of molecular markers can be used for these analyses. Restriction fragment length polymorphisms or RFLP were the first type applied to mapping and for many species, these are still the most often used molecular. Although the advent of RFLP launched the molecular mapping revolution, for species such as human their utility has about run its course. Mapping requires polymorphisms between the individuals being mapped. In humans, an informative RFLP, that is one that maps near a gene of interest, may exist for one population but not be useful for a second population. Therefore the molecular marker was only good in that one population. What was needed was a marker system that was highly polymorphic and recognized many alleles (molecular variants). This extends the usefulness of any one specific marker linked to a gene to many human populations.

One such marker system is variable number tandem repeats (VNTR) or minisattelites. The core of the VNTR is a sequences usually 15-100 base pairs long. What makes these sequences particularly useful is that during evolution the core sequence under went tandem duplication and dispersal throughout the genome. The result is that a core sequence at one chromosomal location may have been repeated ten times, but at another location repeated 20 times. Therefore using a probe recognizing the core sequence allows the researchers to score both locations. In practise, the human genome may have 10 locations to which the core sequence was dispersed, and at each location the number of repeats varies. Thus the term variable number tandem repeat represents the variation in length. The human geneticists then uses pedigree analysis to find associations between a VNTR of a given length and the specific gene they are trying to map.

Microsatellites are another marker system developed for humans and now being applied to other species. This molecular marker is based on dinucleotide repeats. Scatterred among the chromosomes of all species are repeats such as 5'-CA-3' which are repeated a variable number of times at different locations. As with VNTR, a specific size of microsatellite may be linked to a gene of interest. Once this linkage is established, the gene can be followed using the microsatellite.

The primary technical difference when applying these two marker systems is that VNTR are scored used Southern hybridization techniques and microsatellite are analyzed using the polymerase chain reaction. For VNTR a probe to the core sequence must be found. That probe will usually generate a stair step pattern of hybridization, in which the bands differ from the previous band by the length of the core sequence. For micorosatellites, the sequences that border the dinucleotide repeat are conserved. Once the bordering sequence is determined, primers can be designed to initiate the PCR. Because the length of the dinucleotide repeat length varies, products of different length will be obtained. For both systems, geneticists must determine which Southern hybridization band or PCR product is linked to the gene of interest.

Whether RFLP, VNTR or microsatellites are used, a segregating population must be scored. Each of the individuals is analyzed for all of the markers, and two and three point analyses are performed among all two and three combinations of markers. This analysis is exactly analagous to that described for phenotypic genes. In reality, though, analyzing hundreds of markers simulataneously is not feasible by hand. Rather computer programs are used to determine linkages and develop the map. The development of programs such as MAPMAKER, LINKAGE and FASTMAP have greatly facilitated mapping efforts in many species. Today, molecular maps of all major species of interest are available for medical or agricultural scientists. And these scientists are using these maps to isolate genes of interest.

Copyright © 1997. Phillip McClean