Kinds of Assays

One component of the success of the Human Genome Project, in concert with the realization that all diseases have a genetic basis, is the advent of rapid and relatively inexpensive molecular genetic technologies that can be run at high throughput. There are different technologies for different types of mutations, and most are limited to specialized genetic laboratories or cancer research settings. The technologies for detecting mutations in the major cancer susceptibility genes are constantly evolving but can basically be divided into tests of gene function, such as protein truncation tests, gel shift assays, enzymatic mutations screens, and methods to directly sequence the genes.

Because many of the genetic mutations associated with cancer syndromes result in premature truncation of the protein product, protein truncation tests have been widely used. This approach uses in vitro transcription and translation to produce a radiolabeled protein. Truncated forms can be detected when electrophoresed against normal controls on an agarose gel.1 Protein truncation tests are misleading when the gene length is normal, but its function is altered, or when the protein products produced by the mutated gene are too small for detection by this method.2

Gel shift assays compare the mobility through a gel matrix of a test DNA sample to that of a control sample. The motility of DNA in a gel matrix is determined by its length, base composition, single- and double-strand characteristics, and double-strand mobility in the presence of mismatched controls. Examples of gel shift assays are single-strand con formation polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis (HA), and conformation-sensitive gel electrophoresis (CSGE). Although gel shift assays are relatively easy to perform and inexpensive, their sensitivity is lower than other types of assays, making them less appropriate for clinical genetic testing.

Based on the principle that heteroduplexes form between wild-type and mutant genetic sequences, enzymatic mutation detection (EMD) methods use enzymes with high specificity for insertions, deletions, and base-substitution mismatches. Normal and mutant alleles of the target gene are amplified and labeled with fluorescent dyes. The enzyme scans the double-stranded DNA until it detects a structural distortion, where it cleaves the genetic material, forming two shorter, radiolabeled fragments. These products are analyzed on an automated DNA sequencer for relative mobility. EMD is easy to use and highly specific for all types of alterations and has the advantage of detecting multiple sequence variants in the same polymerase chain reaction (PCR) product.2

Direct sequencing of the gene is the gold standard for mutation scanning. All the coding regions, as well as the intron-exon boundaries of a gene, are amplified by PCR and sequenced, either manually or by automated techniques, in 250 to 400 base pairs. This approach is costly and labor intensive. Direct sequencing can miss certain types of mutations or large deletions or can detect mutations of unknown clinical significance. Many of these technical limitations will most likely be eliminated as the technology is improved and as clinical correlations are established for each mutation.

Clinicians considering using a genetic testing facility for clinical purposes should consider the quality control circumstances of the testing facility being considered. All laboratories doing clinical genetic testing should be certified by the Clinical Laboratory Improvement Act (CLIA) and the College of American Pathology (CAP). Access to a medical geneticist is helpful to assist in test interpretation for difficult cases.

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