Analysis of a polyadenine tract of the transforming growth factor-beta type II receptor gene in colorectal cancers by non-gel-sieving capillary electrophoresis.
We developed a method to analyze a polyadenine tract, the (A)10 repeat, within the cysteine-rich domain of the transforming growth factor-beta (TGF-beta) type II receptor gene using a non-gel-sieving capillary electrophoresis technique and applied it to the DNA diagnosis of colorectal cancers. This method consists of single-strand DNA amplification of the (A)10 repeat by an asymmetric PCR technique and capillary electrophoresis. A higher concentration of dATP in the PCR reaction mixture led to more specific amplification of the (A)10 repeat. Under the optimal electrophoretic conditions, one nucleotide difference could be determined in 8 to 32 nucleotides. One or two base deletions of the (A)10 repeat in colorectal cancers could be detected under these conditions within 30 min, and the results coincided with those obtained on DNA sequencing analyses. According to a sensitivity study, we could detect the deleted sequence if it was present in 12.5% or more of the wild-type allele. The reproducibility of this technique was satisfactory because the intraassay imprecision (CV) (n = 10) was 1.4%. These results indicate that capillary electrophoretic analysis of small repeated sequences results in easier handling and more feasible automation, compared with conventional gel electrophoretic analysis.
Oto
M
M
Department of Biotechnology, Tokyo Technical College, Japan. wh6m-ootu@asahi-net.or.jp
Koguchi
A
A
Yuasa
Y
Y
D001322:Autoanalysis; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics; D004273:DNA, Neoplasm / Q000032:analysis; D003838:Deoxyadenine Nucleotides / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D017353:Gene Deletion; D006801:Humans; D018895:Microsatellite Repeats; D011061:Poly A / Q000032:analysis*; D016133:Polymerase Chain Reaction; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D004273:DNA, Neoplasm; D003838:Deoxyadenine Nucleotides; D018125:Receptors, Transforming Growth Factor beta; D011061:Poly A; C026600:2'-deoxyadenosine triphosphate