Practical application of fluorescence-based image analyzer for PCR single-stranded conformation polymorphism analysis used in detection of multiple point mutations.
We present a practical application of polymerase chain reaction single-strand conformational polymorphism (PCR-SSCP) using a fluorescence-based image analyzer. Fluorescence-labeled primers were used to amplify target sequences. After nondenaturing polyacrylamide gel electrophoresis using a conventional sequencing gel apparatus, the glass plate was directly transferred from the gel box to the image analyzer. High-quality computer storage of the imaging data allowed the image intensity to be quantified over a wide range. Various data bases can be constructed on the basis of this imaging data. This system is suitable for use in clinical examination settings, and its application, to detect ras gene mutations in human pancreatic cancer specimens, is presented.
Takahashi-Fujii
A
A
Biotechnology Research Laboratories, Shiga, Japan.
Ishino
Y
Y
Shimada
A
A
Kato
I
I
D001483:Base Sequence; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics*; D005453:Fluorescence; D011905:Genes, ras; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010190:Pancreatic Neoplasms / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic
D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded