- The haplotype is the particular combination of alleles in a defined region of some chromosome, in effect the genotype in miniature. Originally used to described combinations of MHC alleles, it now may be used to describe particular combinations of RFLPs, SNPs, or other markers.
- DNA fingerprinting analyzes the differences between individuals of the fragments generated by using restriction enzymes to cleave regions that contain short repeated sequences. Because these are unique to every individual, the presence of a particular subset in any two individuals can be used to define their common inheritance (e.g. a parent-child relationship).
- RFLPs and SNPs can be the basis for linkage maps and are useful for establishing parent-progeny relationships.
Recombination frequency can be measured between a restriction marker and a visible phenotypic marker as illustrated in Figure 3.3. So a genetic map can include both genotypic and phenotypic markers.
Because restriction markers are not restricted to those genome changes that affect the phenotype, they provide the basis for an extremely powerful technique for identifying genetic loci at the molecular level. A typical problem concerns a mutation with known effects on the phenotype, where the relevant genetic locus can be placed on a genetic map, but for which we have no knowledge about the corresponding gene or protein. Many damaging or fatal human diseases fall into this category. For example cystic fibrosis shows Mendelian inheritance, but the molecular nature of the mutant function was unknown until it could be identified as a result of characterizing the gene.
If restriction polymorphisms occur at random in the genome, some should occur near any particular target gene. We can identify such restriction markers by virtue of their tight linkage to the mutant phenotype. If we compare the restriction map of DNA from patients suffering from a disease with the DNA of normal people, we may find that a particular restriction site is always present (or always absent) from the patients.
A hypothetical example is shown in Figure 3.4. This situation corresponds to finding 100% linkage between the restriction marker and the phenotype. It would imply that the restriction marker lies so close to the mutant gene that it is never separated from it by recombination.
The identification of such a marker has two important consequences:
- It may offer a diagnostic procedure for detecting the disease. Some of the human diseases that are genetically well characterized but ill defined in molecular terms cannot be easily diagnosed. If a restriction marker is reliably linked to the phenotype, then its presence can be used to diagnose the disease.
- It may lead to isolation of the gene. The restriction marker must lie relatively near the gene on the genetic map if the two loci rarely or never recombine. Although "relatively near" in genetic terms can be a substantial distance in terms of base pairs of DNA, nonetheless it provides a starting point from which we can proceed along the DNA to the gene itself.
The frequent occurrence of SNPs in the human genome makes them useful for genetic mapping. From the 1.4 × 106 SNPs that have already been identified, there is on average an SNP every 1-2 kb. This should allow rapid localization of new disease genes by locating them between the nearest SNPs (Sachidanandam et al., 2001).
On the same principle, RFLP mapping has been in use for some time. Once an RFLP has been assigned to a linkage group, it can be placed on the genetic map. RFLP mapping in man and mouse has led to the construction of linkage maps for both genomes. Any unknown site can be tested for linkage to these sites and by this means rapidly placed on to the map (Donis-Keller et al., 1987; Dietrich et al., 1996; Dib et al., 1996). Because there are fewer RFLPs than SNPs, the resolution of the RFLP map is in principle more limited.
The frequency of polymorphism means that every individual has a unique constellation of SNPs or RFLPs. The particular combination of sites found in a specific region is called a haplotype, a genotype in miniature. Haplotype was originally introduced as a concept to describe the genetic constitution of the major histocompatibility locus, a region specifying proteins of importance in the immune system (see 25 Immune diversity). The concept now has been extended to describe the particular combination of alleles or restriction sites (or any other genetic marker) present in some defined area of the genome.
The existence of RFLPs provides the basis for a technique to establish unequivocal parent-progeny relationships. In cases where parentage is in doubt, a comparison of the RFLP map in a suitable chromosome region between potential parents and child allows absolute assignment of the relationship. The use of DNA restriction analysis to identify individuals has been called DNA fingerprinting. Analysis of especially variable "minisatellite" sequences is used mapping in the human genome (for review see White et al., 1985; Gusella, 1986) (see 4.14 Minisatellites are useful for genetic mapping).