Detection of the major mutation M467T causing cystinuria by single-strand conformation polymorphism analysis using capillary electrophoresis.
We present a fast detection of M467T, the major mutation causing cystinuria, by capillary electrophoresis version of single-strand conformation polymorphism (SSCP). The DNA fragment (317 bp) carrying the point mutation was amplified by polymerase chain reaction (PCR) on the exon 8 of the SLC3A1 gene, which encodes for the transmembrane glycoprotein rBAT, a part of the active cystine and dibasic amino acids transporter. The complementary strands of the fragment were labeled by fluorescein and TAMRA, respectively. Thus, the electromigration of both strands was recorded independently as a laser-induced fluorescence (LIF) signal, what enabled an effective optimization of separation conditions. The injected sample was denatured by immersing the inlet of the separation capillary into a vial with 0.1 M solution of NaOH prior to analysis. Under optimum conditions, the SSCP analysis in poly(vinyl alcohol) (PVA)-coated silica-fused capillary of an effective length of 15 cm, filled with 4% linear polyacrylamide (LPA) solution, was accomplished in approximately 6 min. The experimentally observed mobility shifts of single-stranded DNA (ssDNA) fragments were compared to the appearance of their calculated two-dimensional conformations using Version 3.0 of MFOLD software. The number of nucleotides involved in the duplex regions of theoretical structures correlates well with their real migration order in the sieving medium.
Klepárník
Karel
K
Institute of Analytical Chemistry, Czech Academy of Sciences, Brno, Czech Republic. klep@iach.cz
Grochová
Diana
D
Skopková
Zuzana
Z
Adam
Tomás
T
D026923:Amino Acid Transport Systems, Basic; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D003555:Cystinuria / Q000175:diagnosis* / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D005091:Exons; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D020125:Mutation, Missense; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D017433:Protein Structure, Secondary
D026923:Amino Acid Transport Systems, Basic; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; C499489:SLC7A9 protein, human