Techniques for Genetic Testing

Chromosomal analysis

Down syndrome (trisomy 21) and other abnormalities in chromosomal number and gross structure may be definitively diagnosed by chromosomal analysis (cytogenetics). This technique requires culture of live cells obtained from amniocentesis or chorionic villus sampling and therefore takes some time to complete.

Cells in culture are allowed to enter metaphase and then the condensed chromosomes are extracted and stained. The chromosomes are photographed and identified visually by their size, configuration and banding (staining) pattern. Matching chromosomes are paired, and extra chromosomes or malformed chromosomes are identified visually.

Fluorescent in-situ hybridization

A new technique called FISH (fluorescent in-situ hybridization) promises to speed diagnosis in some cases and minimize the amount of tissue required. In this technique, fluorescently-labeled molecular probes to specific genes on a chromosome are applied to individual cells and allow visualization of portions of that chromosome on a cell-by-cell basis. This technique requires the DNA sequences of interest to be known so that appropriate probes may be produced; however, it has the advantage that it can be used directly on relatively few interphase cells, and doesn't require cell culture. It also has the capability to detect and analyze fetal cells that escape into the maternal circulation, which could decrease the need for amniocentesis.

Direct analysis with DNA probes

 In the near future, as the normal human genome is sequenced and more abnormal gene sequences become known, direct analysis for abnormal genes using DNA probes will be more widely used. In this technique, DNA is prepared from cells, fragmented with restriction enzymes, and separated using gel electrophoresis. Nucleic acid probes with sequences complementary to known genetic abnormalities identify the presence of the abnormal sequences in the test DNA. Click the shadowed text in the example below for a brief overview of this technique (there are several sequences--be sure to continue to click the shadowed text until the end). A related method currently in development automates analysis with DNA probes by attaching millions of probes to a silicon chip similar to a computer chip. The chip contains numerous different probes arranged in a pattern across its surface. Patient DNA is hybridized to the chip sequences, then second probes with fluorescent tags are hybridized to the free ends of the patient's DNA fragments. A sensitive photomultiplier reads the pattern of fluorescence on the chip and can diagnose multiple genetic disorders in a single pass. The chip is reusable and offers the possibility of high speed, automated DNA analysis.

Restriction fragment length polymophism (RFLP)

 The technique described above requires that the abnormal sequence be known. Currently, most abnormalities have not been sequenced. In other cases, problems may result from multiple abnormalities, only some of which are known. These unknown genetic defects are detectable using a technique called RFLP analysis.

RFLP analysis takes advantage of the fact that changes in gene sequences change the locations that restriction enzymes cut DNA (because these enzymes are sequence-specific). DNA with abnormal sequences will yield a different mixture of fragment lengths after enzyme digestion than normal DNA, thus the name "fragment length polymorphism."

If a known sequence can be found that is close to a gene of interest, then, for some restriction enzymes, an abnormality in the gene is likely to change the length of the fragment that contains the known sequence. When the DNA fragment mix is separated on a gel, the known sequence will appear in an unexpected spot because the length of its fragment is different. Click the shadowed text below for a brief overview of this technique.

Last modified: 1/22/97; Author: J. Harrison