Clinical Chemistry
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Clinical Chemistry 43: 759-763, 1997;
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(Clinical Chemistry. 1997;43:759-763.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Analysis of a polyadenine tract of the transforming growth factor-ß type II receptor gene in colorectal cancers by non-gel-sieving capillary electrophoresis

Michiei Oto1,2,a, Akira Koguchi3 and Yasuhito Yuasa2

1 Department of Biotechnology, Tokyo Technical College, 1-15-5 Higashi, Kunitachi-shi, Tokyo 186, Japan.

2 Department of Hygiene and Oncology, Tokyo Medical and Dental University School of Medicine, Yushima, Bunkyo-ku, Tokyo 113, Japan.

3 Nippon Bio-Rad Laboratories, Higashi-Nippori, Arakawa-ku, Tokyo 116, Japan.
a Author for correspondence. Fax 81-425-74-0184; e-mail wh6m-ootu{at}asahi-net.or.jp

We developed a method to analyze a polyadenine tract, the (A)10 repeat, within the cysteine-rich domain of the transforming growth factor-ß (TGF-ß) 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.


Key Words: indexing terms: polymerase chain reaction • DNA diagnosis




The following articles in journals at HighWire Press have cited this article:


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 Clin. Chem.Home page
C. Gelfi, M. Perego, P. G. Righetti, S. Cainarca, S. Firpo, M. Ferrari, and L. Cremonesi
Rapid capillary zone electrophoresis in isoelectric histidine buffer: high resolution of the poly-T tract allelic variants in intron 8 of the CFTR gene
Clin. Chem., May 1, 1998; 44(5): 906 - 913.
[Abstract] [Full Text] [PDF]


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Copyright © 1997 by the American Association for Clinical Chemistry.