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From Sample to Result: Fragile X DNA testing is one of the more complex molecular genetic tests to perform and interpret. The procedure involves many steps performed over several days and requires a high degree of technical expertise. There is a wide variety of possible results, and interpretation requires experience. The time from the arrival of the sample in the laboratory to the completion of the written report is usually 10-14 days. Sample Log-in After the sample reaches the lab, it is given a number for tracking and is logged into the patient database or accession notebook. All information about the sample is recorded and a patient folder is created to hold all paperwork relating to the case. Isolation of DNA The first step in the laboratory is to isolate DNA (genetic material) from the sample. Blood samples: Red blood cells (which do not contain DNA) are removed by adding a solution which causes them to burst. The white blood cells are then collected at the bottom of the tube and disrupted to release their DNA. The DNA is purified away from all other cellular material and in the last step it materializes as long white strands. These strands are dissolved in a small amount of water and the resulting clear, viscous liquid is the DNA used for testing. Amniocytes or chorionic villus cells: The DNA isolation approach for prenatal samples is very similar to that for blood except that there are no red blood cells to remove and usually fewer cells are available. DNA analysis There are two separate approaches to fragile X DNA testing, Southern blot analysis and PCR analysis, described below. They have different advantages. Southern blot analysis is the method of choice for identifying full mutations and large premutations and determining if the gene is methylated while PCR analysis allows accurate determination of CGG repeat number for normal, grey zone and premutation alleles. Fragile X testing is most accurate and reliable when both approaches are used in the laboratory. A recent study of labs offering fragile X testing indicated that about 38% of those surveyed do not perform PCR analysis. As with many DNA tests, fragile X DNA analysis utilizes the fact that DNA is double stranded and one strand of a gene binds specifically to the other. The FMR1 gene of a person being tested can be 'seen' by sticking onto it a purified piece of the FMR1 gene with an attached radioactive label. This piece of gene is called a 'probe' and the mild radioactive signal the label emits allows the patient's bound gene to be visualized by exposure to X-ray film. Southern blot analysis: A portion of the DNA isolated from the patient sample is cut into pieces of many sizes using certain enzymes which recognize and cleave at specific DNA sequences. Among the millions of DNA fragments that result are some containing the section of the FMR1 gene with the CGG repeat. When there is no mutation, these FMR1 fragments are of a single known size. If a mutation is present, the fragments are longer. Furthermore, fragments from FMR1 genes that are methylated can be distinguished by size from those that are unmethylated because one of the enzymes utilized fails to cut if methylation is present. All the DNA fragments in the mixture are separated according to size by using an electric current to draw them through a porous rubbery material (made from a component of seaweed) called a gel. Each patient DNA sample is applied to a slot at the top of the gel and runs down a narrow strip of the gel. Short fragments move faster and further than long fragments. The resulting long smear of DNA fragments can be visualized by staining with a dye that glows bright orange. At this point the FMR1 fragments are indistinguishable from all the other DNA fragments. The smears of DNA fragments are transferred onto a thin sheet of nylon by blotting and then the sheet is bathed with a radioactively-labeled FMR1 gene probe. The probe singles out the matching FMR1 gene fragments and binds tightly. Exposure of the nylon sheet to X-ray film results in short black horizontal lines on the film corresponding to the positions of the patient's FMR1 gene fragments. These lines ('bands') create a vertical pattern that is different depending on the presence or absence of a mutation, the type of mutation, the methylation status and the sex of the patient. Some examples of these patterns are shown in the diagram entitled 'Fragile X Analysis by Southern Blot' at the end of this article. Many unusual patterns not illustrated here can also be encountered. Caution is required when interpreting a pattern suggesting a small premutation. Slight separation of two unmethylated bands may be seen when two normal alleles or a normal and a 'grey zone' allele differ significantly in size. Precise CGG repeat number determination for premutation and grey zone alleles requires PCR analysis. PCR analysis: PCR (polymerase chain reaction) analysis for fragile X involves generating a million copies of a short section of the patient's FMR1 gene containing the CGG repeat. The resulting pieces of DNA (known as PCR products) are much shorter than those analyzed by Southern blot and much smaller size differences can be detected. After separation on a gel, pieces differing in length by as little as one CGG repeat can be distinguished. This is why quite accurate determination of CGG repeat number for normal alleles and premutations is possible by PCR analysis. An effective way of visualizing the FMR1 PCR product(s) is to use a probe that binds to the CGG repeat stretch. X-ray film exposure results in one, two or no bands. The diagram entitled 'Fragile X Analysis by PCR' at the end of this article shows the appearance of these bands and gives several examples of results. As a rule, PCR analysis does not reliably detect full mutations, so absence of a band is suggestive of a full mutation in a male. Presence of the full mutation must be confirmed by Southern blot analysis. For a female with a full mutation, the PCR result looks the same (a single band) as for a female with two normal alleles of the same CGG repeat number. Again, complementary results from Southern blot analysis are essential. Annette K. Taylor, M.S., Ph.D. |
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