Genetic mutation analysis by CE with LIF detection using inverse-flow derivatization of DNA fragments.
Inverse-flow derivatization is a novel approach to obtain fluorescent DNA derivatives in DNA analysis based on CE with LIF detection. In the present work, we want to explore the feasibility of the application of this method into the mutation detection based on constant denaturant capillary electrophoresis (CDCE) and SSCP analysis. The DNA fragments were first amplified by PCR using a pair of common primers without fluorescent label, and then the mutations were determined by CDCE or SSCP analysis based on CE-LIF with inverse-flow derivatization of DNA fragments. The experimental conditions were investigated systematically, and different labeling modes including inverse-flow derivatization, on-column derivatization and fluorescent labeled primer technique were compared. The inverse-flow derivatization was successfully used in the detection of C677T mutation in the methylenetetrahydrofolate reductase gene by CDCE or SSCP analysis. Our preliminary results demonstrate that inverse-flow derivatization is very simple, inexpensive and sensitive and well suitable for the genetic analysis in clinical diagnosis.
Sang
Fuming
F
College of Chemistry and Chemical Engineering, Shanghai Jiaotong University, Shanghai, P. R. China.
Ren
Haixia
H
Ren
Jicun
J
D004247:DNA / Q000737:chemistry; D004252:DNA Mutational Analysis / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007834:Lasers; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics; D009154:Mutation; D009691:Nucleic Acid Denaturation; D013050:Spectrometry, Fluorescence / Q000379:methods*
D005456:Fluorescent Dyes; D004247:DNA; D042965:Methylenetetrahydrofolate Reductase (NADPH2)