Detection of point mutations by non-isotopic single strand conformation polymorphism

Abstract

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

Mutations linked to familial hypokalaemic periodic paralysis in the calcium channel α1 subunit gene (Cav1·1) are not associated with thyrotoxic hypokalaemic periodic paralysis

“…They were loaded onto a 1·5 mm × 19 cm × 20 cm 7·5% polyacrylamide:bisacrylamide (49 : 1) gel, containing 10% glycerol, and electrophoresed at room temperature for 6 h (28 mA on a constant current) or until the bromophenol blue dye reached the bottom of the gel. After the run, the gel was soaked into an ethanol (10%)/acetic acid (0·5%) solution for 30 min, stained with silver nitrite solution (1%) for 10 min, washed in water and revealed by NaOH (1·5%)/formaldehyde (0·3%) (Pinheiro et al. , 1999).…”

Analysis of the p53 gene and papillomavirus detection in smears from cervical lesions

“…The PCR/SSCP method has high sensitivity and specificity for the detection of mutations comparable to DNA sequencing. 4 P53 alterations are not frequent in cervical cancer, being generally detected in negative HPV tumors. 5 However, we found two cases simultaneously positive for p53 mutation and high-risk HPV infection.…”

“…PCR products were analyzed on 1.3% agar gel with ethidium bromide staining. 2 PCR amplification of p53 exons 5 to 8 was carried out for SSCP analysis as previously described by Pinheiro et al (1999).…”

Paralisia periódica hipocalêmica tirotóxica, uma urgência endócrina: revisão do quadro clínico e genético de 25 pacientes

“…As amostras denaturadas eram corridas em gel de poliacrilamida denaturante a 7,5% contendo glicerol a 10%. Após eletroforese em temperatura ambiente, o gel era fixado em etanol e ácido acético (10%/0,5%) e corado em solução de nitrato de prata a 1% e, posteriormente, revelado com hidróxido de sódio e formaldeido a 1,5% e 0,3%, respectivamente (14). A triagem dos produtos de PCR por meio da técnica de CSGE era preparada usando-se 5µL do produto de PCR aquecido a 95°C por 5mim, mantido a 68°C por mais 1h.…”