Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 51
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Some differences in nucleotide sequence are detected by means of a type of enzyme called arestriction endonuclease, which cleaves double-stranded DNA molecules wherever a particular, short sequence ofbases is present. For example, the restriction enzyme EcoRI cleaves DNA wherever the sequence GAATTCappears in either strand, as illustrated in Figure 4.17.
Restriction enzymes will be considered in detail in Chapter 5.For now, we simply note that their significance is related to the fact that a difference in DNA sequence thateliminates a cleavage site can be detected because the region lacking the cleavage site will be cleaved into onelarger fragment instead of two smaller ones (Figure 4.18).
More rarely, a mutation in DNA sequence will create anew site rather than destroy one already present. Organisms, including human beings, frequently have minordifferences in DNA sequence that are present in homologous, and otherwise completely identical, regions of DNA;any difference that alters a cleavage site will also change the length of the DNA fragments produced by cleavagewith the corresponding restriction enzyme. The different DNA fragments can be separated by size by an electricfield in a supporting gel and detected by various means.
Differences in DNA fragment length produced by thepresence or absence of the cleavage sites in DNA molecules are known as restriction fragment lengthpolymorphisms (RFLPs).RFLPs are typically formed in one of two ways. A mutation that changes a base sequence may result in loss or gainof a cleavage site that is recognized by the restriction endonuclease in use. Figure 4.19A gives an example. On theleft is shown the relevant region in the homologous DNA molecules in a person who is heterozygous for such asequence polymorphism.
The homologous chromosomes in the person are distinguished by the letters a and b. Inthe region of interest, chromosome a contains two cleavage sites and chromosome b contains three. On the right isshown the position of the DNA fragments produced by cleavage after separation in an electric field. Each fragmentappears as a horizontal band in the gel. The fragment from chromosome a migrates more slowly than those fromchromosome b because it is larger, and larger fragments move more slowly through the gel. In this example, DNAfrom a person heterozygous for the a and b types of chromosomes would yield three bands in a gel.
Similarly,Figure 4.17The restriction enzyme EcoRI cleaves double-stranded DNA wherever thesequence 5'-GAATTC-3' is present. In the example shown here, the DNAmolecule contains three EcoRI cleavage sites, and it is cleaved at each site,producing a number of fragments.Page 148Figure 4.18A minor difference in the DNA sequence of two molecules can be detected if the difference eliminates a restrictionsite. (A) This molecule contains three restriction sites for EcoRI, including one at each end. It iscleaved into two fragments by the enzyme. (B) This molecule has a mutated base sequencein the EcoRI site in the middle.
It changes 5'-GAATTC-3' into 5'-GAACTC-3', which is no longercleaved by EcoRI. Treatment of this molecule with EcoRI results in one larger fragment.DNA from homozygous aa would yield one band, and that from homozygous bb would yield two bands.A second type of RFLP results from differences in the number of copies of a short DNA sequence that may berepeated many times in tandem at a particular site in a chromosome (Figure 4.19B). In a particular chromosome,the tandem repeats may contain any number of copies, typically ranging from ten to a few hundred.
When a DNAmolecule is cleaved with a restriction endonuclease that cleaves at sites flanking the tandem repeat, the size of theDNA fragment produced is determined by the number of repeats present in the molecule. Figure 4.19B illustrateshomologous DNA sequences in a heterozygous person containing one chromosome a with two copies of the repeatand another chromosome b with five copies of the repeat. When cleaved and separated in a gel, chromosome ayields a shorter fragment than that from chromosome b, because a contains fewer copies of the repeat.
An RFLPresulting from a variable number of tandem repeats is called a VNTR. The utility of VNTRs in human geneticmapping derives from the very large number of alleles that may be present in the human population. The largenumber of alleles also implies that most people will be heterozygous, so their DNA will yield two bands uponcleavage with the appropriate restriction endonuclease. Because of their high degree of variation among people,VNTRs are also widely used in DNA typing in criminal investigations (Chapter 15).In genetic mapping, the phenotype of a person with respect to an RFLP is a pattern of bands in a gel. As with anyother type of gene, the genotype of a person with respect to RFLP alleles is inferred, insofar as it is possible, fromthe phenotype.
Linkage between different RFLP loci is detected through lack of independent assortment of thealleles in pedigrees, and recombination and genetic mapping are carried out using the same principles that apply inother organisms except that, in human beings, because of the small family size, different pedigrees are pooledtogether for analysis. Primarily through the use of RFLP and VNTR polymorphisms, genetic mapping in humanshas progressed rapidly.A three-generation pedigree of a family segregating for several alleles at a VNTR locus is illustrated in Figure4.20. In this example, each of the parents is heterozygous, as are all of the children.
Yet every person can beassigned his or her genotype because the VNTR alleles are codominant. At present, DNA polymorphisms are theprincipal types of genetic markers used inPage 149Figure 4.19Two types of genetic variation that are widespread in most natural populations of animals and plants.(A) RFLP (restriction fragment length polymorphism), in which alleles differ in the presence or absenceof a cleavage site in the DNA.
The different alleles yield different fragment lengths (shown inthe gel pattern at the right) when the molecules are cleaved with a restriction enzyme. (B) VNTR(variable number of tandem repeats), in which alleles differ in the number of repeating units presentbetween two cleavage sitesgenetic mapping in human pedigrees. Such polymorphisms are prevalent, are located in virtually all regions of thechromosome set, and have multiple alleles and so yield a high proportion of heterozygous genotypes.
Furthermore,only a small amount of biological material is needed to perform the necessary tests. Many of the polymorphismsthat are most useful in genetic mapping result from variation in the number ofFigure 4.20Human pedigree showing segregation of VNTR alleles. Six alleles (1–6) are present in thepedigree, but any one person can have only one allele (if homozygous) or two alleles (ifheterozygous).Page 150tandem copies of a simple repeating sequence present in the DNA, such as5'-. .
. TGTGTGTGTG. . . -3'A simple-sequence polymorphism of this type is called a simple tandem repeat polymorphism, or STRP. Theutility of STRPs in genetic mapping derives from the large number of alleles present in the population and the highproportion of genotypes that are heterozygous for two different alleles.The present human genetic map is based on more than 5000 genetic markers, primarily STRPs, each heterozygousin an average of 70 percent of people tested. Because there is more recombination in females than in males, thefemale and male genetic maps differ in length. The female map is about 4400 cM, the male map about 2700 cM.Averaged over both sexes, the length of the human genetic map for all 23 pairs of chromosomes is about 3500 cM.Because the total DNA content per haploid set of chromosomes is 3154 million base pairs, there is, very roughly, 1cM per million base pairs in the human genome.4.5—Mapping by Tetrad AnalysisIn some species of fungi, each meiotic tetrad is contained in a sac-like structure called an ascus and can berecovered as an intact group.
Each product of meiosis is included in a reproductive cell called an ascospore, and allof the ascospores formed from one meiotic cell remain together in the ascus (Figure 4.21). The advantage of usingthese organisms to study recombination is the potential for analyzing all of the products from each meioticdivision.
Two other features of the organisms are especially useful for genetic analysis: (1) They are haploid, sodominance is not a complicating factor because the genotype is expressed directly in the phenotype; and (2) theyproduce very large numbers of progeny, making it possible to detect rare events and to estimate their frequenciesaccurately.The life cycles of these organisms tend to be short. The only diploid stage is the zygote, which undergoes meiosissoon after it is formed; the resulting haploid meiotic products (which form the ascospores) germinate to regeneratethe vegetative stage (Figure 4.22). In some species, each of the four products of meiosis subsequently undergoes amitotic division, with the result that each member of the tetrad yields a pair of genetically identical ascospores. Inmost of the organisms, the meiotic products, or their derivatives, are not arranged in any particular order in theascus.
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