A simple method for non-radioactive PCR-SSCP using MDE gel solution and a midi gel format: application for the detection of variants in the GLUT1 and CTLA-4 genes.
The need to identify disease-causing mutations and DNA polymorphisms has increased with the continuing identification of new candidate genes. PCR single-strand conformation polymorphism (PCR-SSCP) is one of the techniques most widely used to identify a mutant sequence or a polymorphism in a known gene. However, the original SSCP protocols using the incorporation of radioactive label and polyacrylamide gel electrophoresis on sequencing gels for detection were labour intensive and time-consuming. Here we describe a simple SSCP protocol using MDE gel solution and a midi gel format to detect SSCP variations in the glucose transporter gene GLUT1, that we have previously analysed with the standard radioactive SSCP protocol, and we have also tested this method on the previously described point mutation (A/G transition in exon 1) of the CTLA-4 (cytotoxic T lymphocyte associated-4) gene. All known variants were detected. Based on the results, this technique appears to be simple, with no use of radioactive labels and with easy handling of the gel. Furthermore, it needs little optimisation, is relatively rapid and highly sensitive. We propose this method for the first screening for candidate gene variants.
Sentinelli
F
F
Department of Clinica Science, Institute II Clinica Medica, Division of Endocrinology, University of Rome 'La Sapienza', 00161, Rome, Italy.
Lovari
S
S
Vitale
M
M
Giorgi
G
G
Di Mario
U
U
Baroni
M G
MG
D000069594:Abatacept; D000180:Acrylic Resins; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000235:genetics*; D060908:CTLA-4 Antigen; D004586:Electrophoresis / Q000379:methods*; D051272:Glucose Transporter Type 1; D018796:Immunoconjugates; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D012996:Solutions
D000180:Acrylic Resins; D015703:Antigens, CD; D000943:Antigens, Differentiation; D060908:CTLA-4 Antigen; D051272:Glucose Transporter Type 1; D018796:Immunoconjugates; D009004:Monosaccharide Transport Proteins; D012996:Solutions; C016680:polyacrylamide gels; D000069594:Abatacept