Application of capillary nongel sieving electrophoresis for gene analysis.
Capillary electrophoresis (CE) has proved to be a strong tool for DNA analysis and has found abundant applications in the fields of restriction fragment sizing, mutation screening, polymerase chain reaction (PCR) product characterizing and forensic identifying. CE may be the main alternative to slab gel electrophoresis. Capillary nongel electrophoresis is the most favorable mode when aiming for this purpose because of its advantages of long lifetime, easy operation, good reproducibility, and low expense. In this paper, a new kind of sieving matrix, with mannitol as the additive for capillary electrophoresis, as well as related methods and their application for gene analysis were reported. Nine DNA fragments amplified by multiplex PCR from a normal dystrophin gene were well separated by this system. Three different deletions were found in Duchenne muscular dystrophy (DMD) patients. Three to four copies of the sex-determination region of the Y chromosome (SRY) gene, as well as the phenylalanine hydroxylase (PAH) gene, could be detected in mixed samples. The frequencies of short tandem repeats (STR) in PAH genes was analyzed in 61 normal Chinese individuals and 6 phenylketonuria families. One case of prenatal gene diagnosis was performed. By using this matrix, CE coupled with reverse transcription PCR (RT-PCR), the analysis of the alternative splicing expression pattern of the fragile X mental retardation 1 (FMR1) gene in adult lung tissue was achieved.
Shen
Y
Y
Institute of Basic Medical Sciences, National Laboratory of Medical Molecular Biology, CAMS & PUMC, Beijing, PR, China. sheny@cdm.imicams.ac.cn
Xu
Q
Q
Han
F
F
Ding
K
K
Song
F
F
Fan
Y
Y
Zhu
N
N
Wu
G
G
Lin
B
B
D000328:Adult; D017398:Alternative Splicing; D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D005333:Fetus; D017353:Gene Deletion; D005821:Genetic Techniques; D005838:Genotype; D006801:Humans; D008297:Male; D008353:Mannitol; D009136:Muscular Dystrophies / Q000235:genetics; D010651:Phenylalanine Hydroxylase / Q000235:genetics; D010661:Phenylketonurias / Q000175:diagnosis / Q000235:genetics; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019849:Sex Determination Processes; D020080:Tandem Repeat Sequences; D014998:Y Chromosome
D008353:Mannitol; D010651:Phenylalanine Hydroxylase