An automated fluorescent single strand conformation polymorphism technique for high throughput mutation screening.
OBJECTIVE
To develop a high throughput mutational detection method by multiple fluorescence-labeled polymerase chain reaction (PCR) products.


METHODS
A total of 27 known mutations including 22 substitutions, 3 insertions (1, 2 and 7 bp) and 2 deletions (1 and 2 bp) in the hepatocyte nuclear factor (HNF)-4 alpha, glucokinase and HNF-1 alpha genes were tested. During nested PCR, amplified fragments were labeled with three fluorescent dyes. PCR products were visualized with an ABI-377 fluorescence sequencer using 5% glycerol or 10% sucrose in non-denaturing gel conditions.


RESULTS
Twenty-five of 27 variants (93%) could be detected by combining 5% glycerol and 10% sucrose gel matrix conditions. Twenty-two of 27 (82%) and 18 of 27 (67%) variants were identified using 5% glycerol and 10% sucrose conditions, respectively.


CONCLUSIONS
This fluorescence-based PCR single strand conformation polymorphism technique represents a simple, non-hazardous, time-saving and sensitive method for high throughput mutation detection.
Weng
J
J
Department of Endocrinology, First Affiliated Hospital of Sun Yet-San University of Medical Sciences, Guangzhou 510080, China. gzwengjp@public.guangzhou.gd.cn
Lehto
M
M
Berglund
A
A
Groop
L C
LC
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D005941:Glucokinase / Q000235:genetics; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins; D010750:Phosphoproteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D014157:Transcription Factors / Q000235:genetics
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D005941:Glucokinase