Limitations in the use of SSCP analysis

Fan, Erkang; Levin, D. B.; Glickman, Barry W.; Logan, David M.

doi:10.1016/0027-5107(93)90210-7

Cited by 74 publications

(36 citation statements)

References 10 publications

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“…Our work further confirms a previous report that the resulting SSCP pattern was highly reproducible when conditions were held constant. 30) This was true for all the species studied here. In addition, no modification of SSCP profiles was observed after two rounds of PCR for any of the clones analyzed.…”

Section: Secondary Structuresupporting

confidence: 52%

Single-Strand Conformation Polymorphism for Molecular Variability Studies of Six Viroid Species

Elleuch

Hamdi

Bessaies³

et al. 2013

Bioscience, Biotechnology, and Biochemistry

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Section: Secondary Structuresupporting

confidence: 52%

Single-Strand Conformation Polymorphism for Molecular Variability Studies of Six Viroid Species

Elleuch

Hamdi

Bessaies³

et al. 2013

Bioscience, Biotechnology, and Biochemistry

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“…Several patients with mutations in STAT-1 gene have been recently described; however, they show differences in their clinical presentation. As mentioned before, two kindred with the same heterozygous mutation affecting the dimerization of STAT-1 to form gamma-activated factor were susceptible to mycobacteria but resistant to viruses, with a phenotype similar to that of patients with partial IFN-γR (28). A more recent report described two other children who developed disseminated BCG after vaccination, with remission occurring with antibiotic treatment.…”

Section: Discussionmentioning

confidence: 66%

“…During PCR prior to SSCP analysis some DNA fragments exhibiting a different conformation during electrophoresis might have been generated. The altered electrophoretic mobility could also be a consequence of the thermal and physical conditions of electrophoresis (28). In other cases, the changes in DNA responsible for the altered mobility in the SSCP might be located in the sequence complementary to the primers used during PCR, in regions very difficult to define by the sequencing reaction used.…”

Section: Discussionmentioning

confidence: 99%

Normal expression of IFN-gammaR in four patients with uncommon mycobacterial infection phenotypes

Rugeles

Rincón

Rugeles

et al. 2004

Braz J Med Biol Res

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Several primary immunodeficiency diseases affecting the interleukin 12/interferon gamma (IFN-γ) pathway have been identified, most of them characterized by recurrent and protracted infections produced by intracellular microorganisms, particularly by several species of mycobacteria. In the present study we analyzed the expression of IFN-γ receptor (IFN-γR) and signal transducer and activator of transcription 1 (STAT-1) in 4 children with Mycobacterium tuberculosis infection of uncommon clinical presentation. These molecules were evaluated by flow cytometry and Western blotting in B cells transformed with Epstein-Barr virus and mutations were scanned by single-strand conformational polymorphisms and DNA sequencing. The expression of IFN-γR1 was normal in all 4 patients. The genetic analysis of IFN-γR1 and IFN-γR2 coding sequences did not reveal any mutation. The expression of the STAT-1 molecule was similar in patients and healthy controls; however, when the phosphorylation of this transcription factor in response to IFN-γ activation was evaluated by Western blot, a significant lower signal was evident in one patient. These data indicate that there are no alterations in the expression or function of the IFN-γR chains in these patients. However, the low level of STAT-1 phosphorylation found in one of these patients might be explained by a defect in one of the molecules involved in the signal transduction pathway after IFN-γ interacts with its receptor. In the other three patients the inability to eliminate the mycobacteria may be due to a defect in another effector mechanism of the mononuclear phagocytes.

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“…Although it was first described using radioactive primers ("hot-SSCP"), we used an alternative non-isotopic protocol, considered more sensitive than the original one (19). Although there are many reports using this technique (5,26), it is important to point out that PCR-SSCP has limitations, independently on the protocol adopted, and should be preferentially used to detect point mutations that can be further studied by DNA sequencing methods (9,12).…”

Section: Primers To Amplify Exons 5 6 7 and 8 Of Tp53 Gene And Exonmentioning

confidence: 99%

No mutations found in exons of TP53, H-RAS and K-RAS genes in liver of male Wistar rats submitted to a medium-term chemical carcinogenesis assay

Castelli¹,

Otake²,

Oliveira³

et al. 2002

J. Bras. Patol. Med. Lab.

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The standard protocol to evaluate the carcinogenic potential of chemicals is the long-term bioassay in rodents, not performed in developing countries due to its high cost and complex operational procedures. Our laboratory has established an alternative medium-term bioassay in Wistar rats, also called DMBDD assay, based on the paradigm initiation/promotion of chemical carcinogenesis. This method was accepted by the Brazilian Environment Agency (IBAMA) as an official source of evidence of carcinogenicity. The aim of this study was to evaluate alterations in exons 5 to 8 of the tumor suppressor gene TP53 and exons 1 and 2 of oncogenes K-RAS and H-RAS in neoplastic and preneoplastic hepatic lesions observed in DMBDD assay. The characterization of these alterations may contribute to the recognition of patterns of damage in critical genes, as well as to suggest mechanisms of action of the compounds tested in the protocol. Sixty male Wistar rats were separated into 3 groups: the first was treated with no chemicals; the second received five initiating agents and the third received initiation followed by phenobarbital. Liver DNA samples (obtained from formalin-fixed and paraffin-embedded tissues after histological analysis) were evaluated by the non-isotopic PCR-SSCP technique. No changes in any analyzed exons were detected by the PCR-SSCP banding pattern in all experimental groups. This result suggests that liver mutations in exons 5 to 8 of TP53 and exons 1 and 2 of H-RAS and K-RAS are not among the early molecular alterations occurring in the hepatic carcinogenesis process induced by the DMBDD protocol in male Wistar rats. No mutations found in exons of TP53, H-RAS and K-RAS genes in liver of male Wistar rats submitted to a medium-term chemical carcinogenesis assay Castelli et al.

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Limitations in the use of SSCP analysis

Cited by 74 publications

References 10 publications

Single-Strand Conformation Polymorphism for Molecular Variability Studies of Six Viroid Species

Single-Strand Conformation Polymorphism for Molecular Variability Studies of Six Viroid Species

Normal expression of IFN-gammaR in four patients with uncommon mycobacterial infection phenotypes

No mutations found in exons of TP53, H-RAS and K-RAS genes in liver of male Wistar rats submitted to a medium-term chemical carcinogenesis assay

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