We have developed a procedure for nonradioactive single strand conformation polymorphism analysis and applied it to the detection of point mutations in the human tumor suppressor gene p53. The protocol does not require any particular facilities or equipment, such as radioactive handling, large gel units for sequencing, or a semiautomated electrophoresis system. This technique consists of amplification of DNA fragments by PCR with specific oligonucleotide primers, denaturation, and electrophoresis on small neutral polyacrylamide gels, followed by silver staining. The sensitivity of this procedure is comparable to other described techniques and the method is easy to perform and applicable to a variety of tissue specimens. Key wordsA variety of methods have been proposed that facilitate rapid identification of genetic polymorphisms or somatic cell mutations, among which single strand conformation polymorphism (SSCP) is probably the most widely used. The original SSCP protocol uses radiolabeled oligonucleotides to generate a radioactive PCR product, which is then highly diluted, heat denatured, and analyzed by electrophoresis in a large (40 x 20 cm) nondenaturing gel (1). SSCP is based on the differential sequence-dependent electrophoretic mobilities of single-stranded DNA in nondenaturing polyacrylamide gels: changes in the sequence can cause a shift in the mobility of the analyzed conformers. This method, although sensitive, is both time consuming and cumbersome. There are several reports describing non-isotopic protocols for SSCP analysis (2,3), but these require expensive reagents and equipment such as small-format PhastGel, PhastSystem and Hydrolink-MDE, or the use of composite agarose gels (4).We developed a protocol for the detection of point mutations and deletions by a non-isotopic SSCP analysis on standard silver-stained polyacrylamide gels. The sequences used were PCR amplified regions of the p53 gene from samples of cervical carcinomas, penile carcinomas and gastric carcinomas where point mutations had been previously identified by isotopic SSCP and sequencing.PCR products were generated by specific primers and conditions for exons 5, 6, 7, 8 and 9 of the p53 gene, using the following primers: for exon 5, 5-TACTCCCCTGCC
We report on a case of a patient with HIV infection, diagnosed 18 months prior to the development of an anti-glomerular basement membrane (anti-GBM) rapidly progressive glomerulonephritis; this is probably the first report of such an association. A 30-year-old white man presented with elevation of serum creatinine (1.3 -13.5 mg/dL within one month). At admission, the urinalysis showed proteinuria of 7.2 g/L and 8,000,000 erythrocytes/mL. Renal biopsy corresponded to a crescentic diffuse proliferative glomerulonephritis mediated by anti-GBM, and serum testing for anti-GBM antibodies was positive; antinuclear antibodies (ANA) and anti-neutrophilic cytoplasmic antibodies (ANCA) were also positive. The patient underwent hemodyalisis and was treated with plasmapheresis, cyclophosphamide and prednisone. The association described here is not casual, as crescentic glomerulonephritis is not common in HIV-positive patients, anti-GBM glomerulonephritis is rare and anti-GBM antibodies are frequently observed in HIV-positive subjects when compared to the overall population. Based on the current case and on the elevated frequency of the positivity for such antibodies in this group of patients, it is advisable to be aware of the eventual association between these two conditions and to promote an active search for anti-GBM antibodies and early diagnosis of eventual urinary abnormalities in HIV-positive subjects, considering the severity of anti-GBM glomerulonephritis.
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