PCR protocol for DNA recovery from Spurr's-embedded muscle biopsies

1Department of Public Health and Cell Biology, Tot Vergata University of Rome and C.S.S. Hospital, San Giovanni Rotondo, Rome, Italy; 2Chair of Human Genetics, Catholic University of Rome, Rome, Italy PCR has been used extensively for the retrospective molecular biological studies of a wide variety of pathological archival material, inc luding air-dried or stained bone marrow smears, formalinfixed tissues, fixed cytogenetic preparations, Guthrie spots, and autopsied and frozen tissues. ~1-6) Thus, PCR can be applied to the diagnosis of a variety of genetic disorders that are lethal in early childhood, including medium-chain acylcoenzyme A dehydrogenase deficiency and spinal muscular atrophy, and to the detection of infectious disease pathogens and m i n i m a l residues of disease. C7'8) Spinal muscular atrophies (SMAs; MIM* 253300) are neuromuscular disorders, characterized by proximal, symmetrical l imb and trunk muscle weakness resulting from specific degeneration of anterior horn cells of the spinal cord. (8) SMA I (Werdnig-Hoffman disease), SMAII (an intermediate form), and SMA III (Wohlfart-Kugelberg-Welander disease) are recognized on the basis of age onset, severity, and survival. These three disorders have proved to be a result of allelic mutat ions of a locus assigned to 5q12-q14. ~9) At present, the molecular diagnosis of SMA is performed by linkage analysis using microsatellite DNA markers. (1~ However, linkage analysis is often prevented by the early death of the patient, which in SMA I usually occurs by the age of 18 months . This difficulty can be circumvented by PCR analysis of DNA isolated from microscopic glass slides and/ or frozen muscle tissue of the proband, even on samples stored for several years. (la) A potential difficulty in recovering DNA comes from viscosity of the embedding media. The low-viscosity Spurr's mixture (~2) is widely used in electron microscopy science, where it provides a high-quali ty and rapid infiltration. So far, no protocol has been developed for recovering DNA from Spurr's blocks. Here, we report a reproducible and sensitive method for PCR amplif icat ion of microsatellite DNA from muscle biopsy sections on Spurr's blocks that allows retrospective molecular characterization of deceased patients. Spurr 's-embedded muscle biopsies of the gluteus or vastus of five SMA I patients were prepared 1 m o n t h to 5 yr before use with a commercia l resin kit (Taab Laboratories, Berkshire, England). The mixture, which was composed of vinyl cyclohexane dioxide, diglycidyl ether of polypropylene glycol, and nonenyl succinic anhydride, in a ratio of 10 : 60 : 26 : 0.4, respectively, was combined in a f irm medium, according to the recommendat ions of the manufacturers. Dehydration, infiltration, and polymerizat ion were carried out according to Spurr. (a2) Sections 3-5 m m thick were cut from the blocks using a razor and treated with te trahydrofuran (THF) (Panreac, Barcelona, Spain) at room temperature for 5 rain. Depolymerized material was washed in ether saturated with distilled water to remove THF and dried at room temperature. Fifty microliters of a lysis buffer conta in ing 20 mM Tris-HC1 (pH 8.0), 20 mM EDTA (pH 8.0), 10 mM NaCl, 2% Tween 20, 1 mM dithiothreitol , and 500 i~g/ml proteinase K was added to each sample and incubated overnight at 37~ The lysate was boiled for 8 m i n in a programmable heat ing block (PerkinElmer Roche, Branchburg, NJ) and centrifuged for 10 m i n at 10,000 rpm. Nucleic acids were extracted with phenol / chloroform/isoamylic alcohol solution and precipitated overnight with two volumes of cold absolute e thanol at -20~ DNA was lyophil ized and resuspended in 50 !~1 of a PCR mixture conta in ing 25 pmoles of ol igonucleotide primers; 200 ~I.M each dGTP, dATP, dTTP, and dCTP; 0.5 i~Ci of [c~-32p]dCTP (Amersham International, Amersham, UK); 2.5 units of Taq polymerase (AmpliTaq, PerkinElmer Roche); 50 mM Tris-HC1 (pH 8.5); and 1.5 mM MgCl2, and 0.1% (wt/vol) autoclaved gelatin. Each reaction was overlaid with two drops of minera l oil (Sigma Chemicals, St. Louis, MO). The reactions were performed in a DNA thermocycler (Perkin-Elmer Roche) at 95~ for 5 m i n for one cycle, 95~ for 30 sec, 62~ for 40 sec, 72~ for 1 m i n for 33 cycles, and 72~ for 7 min . On comple t ion of temperature cycling, 5 I~l of loading dye (10 mM NaOH, 95% formamide, 0.05% brompheno l blue, 0.05% xylene cyanol) was added to 4 i~l of amplif ied product and electrophoresed on 8% acrylamide/7 M urea sequencing gels at 40 W. Gels were dried under vacuum and exposed to X-ray fi lm at -80~ with in tens i fy ing screens. This protocol was also successfully applied to the study of six addit ional Spurr 's-embedded specimens stored for 1-8 yr (courtesy of G. Zelano, Catholic University of Rome, Rome, Italy), which were evaluated for the HLADQc~ locus using a commercia l kit (AmpliType, Perkin-Elmer Roche).