About 90% of human genome variation occurs in the form of single nucleotide polymorphisms (SNPs), which are variations involving just one base. These single-base changes can render a restriction site unrecognizable by a specific restriction endonuclease or create a new restriction site. Cleavage with an endonuclease results in fragments of different lengths that can be detected using DNA hybridization. The altered restriction site can be located at the site of a disease-causing mutation or at a site some distance away from the mutation.

Polymorphisms in chromosomal DNA can also arise from the presence of a variable number of tandem repeats (VNTR). These VNTRs are short sequences of DNA repeated in tandem at scattered locations in the genome. The number of repeat units varies from person to person, serving as a unique molecular "fingerprint." Cleavage by restriction enzymes yields fragments of varying lengths depending on the number of repeated segments contained in the fragment. VNTR loci are useful for DNA fingerprint analysis in forensic and paternity cases. It is important to note that these polymorphisms, whether SNP or VNTR, are markers and do not affect the structure, function, or production rate of any specific protein.

When the DNA of an individual gains a restriction site through base substitution, enzymic cleavage produces at least one additional fragment. Conversely, loss of a restriction site due to a mutation results in fewer fragments produced by enzymic cleavage. In individuals heterozygous for a polymorphism, one chromosome has a sequence variation in the DNA while the homologous chromosome does not. Tracing each chromosome from parent to offspring can be done by determining the presence or absence of the polymorphism.

In families with a history of severe genetic disease, prenatal diagnosis along with genetic counseling can help determine the presence of the disorder in a developing fetus. Various diagnostic methods are available with varying sensitivity and specificity. Visualization of the fetus through ultrasound or fetoscopy is useful for genetic abnormalities that cause gross anatomic defects. The chemical composition of amniotic fluid, such as high levels of αfetoprotein, can provide diagnostic clues. Fetal cells obtained from amniotic fluid or chorionic villi biopsy can be used for karyotyping, which assesses the morphology of metaphase chromosomes. Staining and cell sorting techniques allow for rapid identification of trisomies and translocations that involve extra or abnormally long chromosomes. However, molecular analysis of fetal DNA provides the most detailed genetic information.

DNA can be obtained from blood cells, amniotic fluid, or chorionic villi. Previously, growing cells in culture for 2-3 weeks was necessary to obtain sufficient DNA for analysis from amniotic fluid. The ability to amplify DNA through PCR has significantly reduced the time required for DNA analysis.
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.