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Modern Genetic Analysis Chapter 11. Applications of Recombinant DNA Technology
Using Recombinant DNA to Detect Disease Alleles Directly

Recessive mutant phenotypes that follow single-gene inheritance are responsible for more than 500 human genetic diseases. A homozygous person resulting from a marriage between two heterozygotes for the same recessive allele will be affected by the disease. Cells derived from the fetus can be screened at an early enough stage to allow the option of abortion of afflicted fetuses. Many of the enzymes or other proteins that are altered or missing in genetic diseases are now known (refer to the list of inborn errors of metabolism, Table 3-4).

To detect such genetic defects, fetal cells can be taken from the amniotic fluid, separated from other components, and cultured to allow the analysis of chromosomes, proteins, enzymatic reactions, and other biochemical properties. This process, amniocentesis (Figure 11-26), can identify a number of known disorders; Table 11-1 on the following page lists some examples. Chorionic villus sampling (CVS) is a related technique in which a small sample of cells from the placenta is aspirated out with a long syringe. CVS can be performed earlier in the pregnancy than can amniocentesis, which must await the development of a large enough volume of amniotic fluid.

Testing physiological properties or enzymatic activity in cultured fetal cells limits the screening procedure to those disorders that affect characters or proteins expressed in the cultured cells. However, recombinant DNA technology improves the screening for genetic diseases in utero, because the DNA can be analyzed directly. In principle, the gene being tested could be cloned and its sequence compared with that of a cloned normal gene. However, this procedure would be lengthy and impractical, so shortcuts have to be devised to allow more rapid screening. Several of the useful techniques that have been developed for this purpose are explained in this section.

Alteration of Restriction Site by Mutation

Sickle-cell anemia is a genetic disease that is caused by a well-characterized mutational alteration. Affecting approximately 0.25 percent of African Americans, the disease results from an altered hemoglobin, in which the amino acid valine is substituted for glutamic acid at position 6 in the b -globin chain. The GAG-to-GTG change eliminates a cleavage site for the restriction enzyme MstII, which cuts the sequence CCTNAGG (in which N represents any of the four bases). The change from CCTGAGG to CCTGTGG can thus be recognized by Southern analysis with the use of labeled b -globin cDNA as a probe, because the DNA derived from persons with sickle-cell disease lacks one fragment contained in the DNA of normal persons and contains a large (uncleaved) fragment not seen in normal DNA (Figure 11-27).

Probing for Altered Sequences

When a genetic disorder can be attributed to a change in a specific nucleotide, synthetic oligonucleotide probes can identify that change. The best example is a 1-antitrypsin deficiency, which leads to a greatly increased probability of developing pulmonary emphysema. The condition results from a single base change at a known nucleotide position. A synthetic oligonucleotide probe that contains the wild-type sequence in the relevant region of the gene can be used in a Southern blot analysis to determine whether the DNA contains the wild-type or the mutant sequence. At higher temperatures, a complementary sequence will hybridize, whereas a sequence containing even a single mismatched base will not.

PCR Tests

Because PCR allows an investigator to zero in on a potentially defective DNA sequence, it can be used effectively in the diagnosis of diseases in which a specific mutational site is in question. Primers spanning the site are used, and the amplified DNA can be isolated and sequenced or otherwise compared with the wild type.

We have already covered the detection of linked RFLPs as a more indirect diagnosis of the presence of a disease allele.

MESSAGE
Recombinant DNA technology provides sensitive techniques for testing for mutant alleles in people or in embryos in utero.

© 1999 by W. H. Freeman and Company. All rights reserved.