1. Single-base changes: About 90% of human genome variation comes in the form of single nucleotide polymorphisms (SNPs, pronounced “snips”), that is, variations that involve just one base (Fig. 34.14). The substitution of one nucleotide at a restriction site can render the site unrecognizable by a particular restriction endonuclease. A new restriction site can also be created by the same mechanism. In either case, cleavage with an endonuclease results in fragments of lengths that differ from the normal and can be detected by DNA hybridization (see Fig. 34.13). The altered restriction site can be either at the site of a disease-causing mutation (rare) or at a site some distance from the mutation. [Note: The HapMap, developed by The International Haplotype Map Project, is a catalog of common SNP in the human genome. The data are being used in genome-wide association studies (GWAS) to identify those alleles that affect health and disease.] 2. Tandem repeats: Polymorphisms in chromosomal DNA can also arise from the presence of a variable number of tandem repeats (VNTR), as shown in Figure 34.15. These are short sequences of DNA at scattered locations in the genome, repeated in tandem (one after another). The number of these repeat units varies from person to person but is unique for any given individual and, therefore, serves as a molecular “fingerprint.” Cleavage by restriction enzymes yields fragments that vary in length depending on how many repeated segments are contained in the fragment (see Fig. 34.15). Many different VNTR loci have been identified and are extremely useful for DNA fingerprint analysis, such as in forensic and paternity cases. It is important to emphasize that these polymorphisms, whether SNP or VNTR, are simply markers, which, in most cases, have no known effect on the structure, function, or rate of production of any particular protein.
B. Tracing chromosomes from parent to offspring
If the DNA of an individual has gained a restriction site by base substitution, then enzymic cleavage yields at least one additional fragment.
Conversely, if a mutation results in loss of a restriction site, fewer fragments are produced by enzymic cleavage. An individual who is heterozygous for a polymorphism has a sequence variation in the DNA of one chromosome and not in the homologous chromosome. In such individuals, each chromosome can be traced from parent to offspring by determining the presence or absence of the polymorphism.
C. Prenatal diagnosis
Families with a history of severe genetic disease, such as an affected previous child or near relative, may wish to determine the presence of the disorder in a developing fetus. Prenatal diagnosis, in association with genetic counseling, allows for an informed reproductive decision if the fetus is affected.
1.
Methods available: The available diagnostic methods vary in sensitivity and specificity. Visualization of the fetus, for example, by ultrasound or fiberoptic devices (fetoscopy), is useful only if the genetic abnormality results in gross anatomic defects (for example, neural tube defects [NTD]). The chemical composition of the amniotic fluid can also provide diagnostic clues. For example, the presence of high levels of αfetoprotein is associated with NTD. Fetal cells obtained from amniotic fluid or from biopsy of the chorionic villi can be used for karyotyping, which assesses the morphology of metaphase chromosomes. Staining and cell sorting techniques permit the rapid identification of trisomies and translocations that produce an extra chromosome or chromosomes of abnormal lengths. However, molecular analysis of fetal DNA provides the most detailed genetic picture.
2.
DNA sources: DNA may be obtained from blood cells, amniotic fluid, or chorionic villi (Fig. 34.16). For amniotic fluid, it was formerly necessary to grow cells in culture for 2–3 weeks in order to have sufficient DNA for analysis. The ability to amplify DNA by PCR has dramatically shortened the time needed for a DNA analysis.