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Direct inspection of nucleic acids—often called “molecular genetics” or “DNA diagnosis”—is an important tool in a number of clinical areas, including oncology, infectious disease, forensics, and the general study of pathophysiology. A major impact has been in the diagnosis of mendelian disorders. Molecular testing is available for more than 3000 separate hereditary conditions. Once a particular gene is shown to be defective in a given condition, the nature of the pathogenic variant in a patient can be determined by sequencing the nucleotides of the coding exons and the splice sites. One of a variety of techniques can then be used to determine whether that same pathogenic variant is present in other patients with the same disorder. Genetic heterogeneity is so extensive that most mendelian conditions are associated with numerous pathogenic variants at one locus—or often multiple loci—that produce the same phenotype. Pathogenic variants at several hundred different genes cause vitreoretinal disorders, such as retinitis pigmentosa, and changes in several dozen genes cause familial hypertrophic cardiomyopathy. This fact complicates DNA diagnosis of patients and screening for carriers of defects in specific genes.

A few conditions are associated with relatively few pathogenic variants or with only one highly prevalent pathogenic variant. For example, all sickle cell disease is caused by exactly the same change of glutamate to valine at position 6 of beta-globin, and that substitution in turn is due to a change of one nucleotide at the sixth codon in the beta-globin gene. But such uniformity is the exception. In cystic fibrosis, about 70% of heterozygotes of northern European ancestry have an identical deletion of three nucleotides that causes loss of a phenylalanine residue from a chloride transport protein; however, the remaining 30% of pathogenic variants of that protein are diverse (several thousand have been discovered), so that no simple screening test will detect all carriers of cystic fibrosis.

Reviews of the technical status of DNA analysis appear regularly in the medical literature. PCR studies revolutionized many aspects of molecular biology, and DNA diagnosis came to involve this technique. If the sequences of the 10–20 nucleotides at the ends of a region of DNA of interest (such as a portion of a gene) are known, then “primers” complementary to these sequences can be synthesized. When even a minute amount of DNA from a patient (eg, from a few leukocytes, buccal mucosal cells, or hair bulbs) is combined with the primers in a reaction mixture that replicates DNA—and after several dozen cycles are then performed—the region of DNA between the primers will be amplified exponentially. For example, the presence of early HIV infection can be detected after PCR amplification of a portion of the viral genome.

The speed of sequencing DNA has accelerated tremendously, and its cost has plummeted due to the arrival of “next-generation sequencing.” The “Holy Grail” of DNA analysis was defined over a decade ago as the “$1000 human genome,” and that benchmark was reached in 2014. ...

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