October 15, 2012

Minisatellites are useful for genetic mapping


KEY TERMS:
  • Microsatellite DNAs consist of repetitions of extremely short (typically <10 bp) units.
  • Minisatellite DNAs consist of ~10 copies of a short repeating sequence. the length of the repeating unit is measured in 10s of base pairs. The number of repeats varies between individual genomes.
  • VNTR (variable number tandem repeat) regions describe very short repeated sequences, including microsatellites and minisatellites.
  • 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).
KEY CONCEPTS:
  • The variation between microsatellites or minisatellites in individual genomes can be used to identify heredity unequivocally by showing that 50% of the bands in an individual are derived from a particular parent.  

Sequences that resemble satellites in consisting of tandem repeats of a short unit, but that overall are much shorter, consisting of (for example) from 5-50 repeats, are common in mammalian genomes. They were discovered by chance as fragments whose size is extremely variable in genomic libraries of human DNA. The variability is seen when a population contains fragments of many different sizes that represent the same genomic region; when individuals are examined, it turns out that there is extensive polymorphism, and that many different alleles can be found (Jeffreys, Wilson, and Thein, 1985).
The name microsatellite is usually used when the length of the repeating unit is <10 bp, and the name minisatellite is used when the length of the repeating unit is ~10-100 bp, but the terminology is not precisely defined. These types of sequences are also called VNTR (variable number tandem repeat) regions.
The cause of the variation between individual genomes at microsatellites or minisatellites is that individual alleles have different numbers of the repeating unit. For example, one minisatellite has a repeat length of 64 bp, and is found in the population with the following distribution:
7%  18 repeats
11%  16 repeats
43%  14 repeats
36%  13 repeats
4%  10 repeats
The rate of genetic exchange at minisatellite sequences is high, ~104 per kb of DNA. (The frequency of exchanges per actual locus is assumed to be proportional to the length of the minisatellite.) This rate is ~10× greater than the rate of homologous recombination at meiosis, that is, in any random DNA sequence.

The high variability of minisatellites makes them especially useful for genomic mapping, because there is a high probability that individuals will vary in their alleles at such a locus. An example of mapping by minisatellites is illustrated in Figure 4.27. This shows an extreme case in which two individuals both are heterozygous at a minisatellite locus, and in fact all four alleles are different. All progeny gain one allele from each parent in the usual way, and it is possible unambiguously to determine the source of every allele in the progeny. In the terminology of human genetics, the meioses described in this figure are highly informative, because of the variation between alleles.
One family of minisatellites in the human genome share a common "core" sequence. The core is a GC-rich sequence of 10-15 bp, showing an asymmetry of purine/pyrimidine distribution on the two strands. Each individual minisatellite has a variant of the core sequence, but ~1000 minisatellites can be detected on Southern blot by a probe consisting of the core sequence.
Consider the situation shown in Figure 4.27, but multiplied 1000×. The effect of the variation at individual loci is to create a unique pattern for every individual. This makes it possible to assign heredity unambiguously between parents and progeny, by showing that 50% of the bands in any individual are derived from a particular parent. This is the basis of the technique known as DNA fingerprinting.

Both microsatellites and minisatellites are unstable, although for different reasons. Microsatellites undergo intrastrand mispairing, when slippage during replication leads to expansion of the repeat, as shown in Figure 4.28 (Jeffreys et al., 1988). Systems that repair damage to DNA, in particular those that recognize mismatched base pairs, are important in reversing such changes, as shown by a large increase in frequency when repair genes are inactivated (Strand et al., 1993). Because mutations in repair systems are an important contributory factor in the development of cancer, tumor cells often display variations in microsatellite sequences (see 30.29 Defects in repair systems cause mutations to accumulate in tumors).
Minisatellites undergo the same sort of unequal crossing-over between repeats that we have discussed for satellites (see Figure 4.1). One telling case is that increased variation is associated with a meiotic hotspot (Jeffreys, Murray, and Neumann, 1998). The recombination event is not usually associated with recombination between flanking markers, but has a complex form in which the new mutant allele gains information from both the sister chromatid and the other (homologous) chromosome (Jeffreys et al., 1994).
It is not clear at what repeating length the cause of the variation shifts from replication slippage to recombination.