Clinical Chemistry AACC Online Job Center
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 45: 1718-1724, 1999;
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (18)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nakamura, Y.
Right arrow Articles by Ide, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakamura, Y.
Right arrow Articles by Ide, T.
Related Collections
Right arrow Molecular Diagnostics and Genetics
(Clinical Chemistry. 1999;45:1718-1724.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Simple, Rapid, Quantitative, and Sensitive Detection of Telomere Repeats in Cell Lysate by a Hybridization Protection Assay

Yasuhiro Nakamura1, Minoru Hirose2, Hajime Matsuo1, Naohiro Tsuyama1, Keiichi Kamisango2 and Toshinori Ide1,a

1 Department of Cellular and Molecular Biology, Hiroshima University School of Medicine, Kasumi 1-2-3, Hiroshima City, Hiroshima 734-8551, Japan.

2 Diagnostic Science Laboratories, Chugai Diagnostic Science Company Ltd., 3-41-8 Takada, Toshima-ku, Tokyo 171-8545, Japan.
a Author for correspondence. Fax 81-82-257-5294; e-mail tide{at}pharm.hiroshima-u.ac.jp

Background: Detection of telomere repeats by Southern hybridization of genomic DNA is time consuming, and the reading of a mean terminal restriction fragment (TRF) length from a smear pattern of an autoradiogram can be inaccurate. We developed a hybridization protection assay (HPA) for telomere repeats.

Methods: We heated 5 µL of DNA solution or 10 µL of cell or tissue lysate at 95 °C for 5 min, mixed it with 100 µL of hybridization solution containing 3 x 106 relative light units of acridinium ester-labeled probe, and incubated the mixture for 20 min at 60 °C. We then added 300 µL of selection buffer and incubated the mixture for 10 min at 60 °C to differentially hydrolyze unhybridized probe. Chemiluminescence was measured for 2 s per tube.

Results: The amount of telomere repeats was assayed by HPA within linearity from 10 to 3000 ng of purified genomic DNA or from 1000 to 100 000 cell equivalents of lysate. To normalize the amount of DNA in lysate, the amount of Alu sequence was measured by HPA. A ratio of telomere to Alu (TA ratio) = 0.01 corresponded to ~2 kbp of mean TRF length determined by Southern blotting in cultured fibroblast and colorectal tissue samples. The TA ratio decreased from 0.06 to 0.02 with increasing division age from 30 to 90 population doubling levels of cultured human fetal fibroblasts. The assay required ~45 min from collection of cell or tissue samples.

Conclusions: The amount of telomere repeats was quantitatively measured by HPA in 10 ng of sheared genomic DNA or in the lysate of 1000 cells. This method is simple, rapid, quantitative, sensitive, and applicable to the measurement of telomere repeats in clinical samples such as needle biopsy specimen or as few as 1000 cells in body fluid or washings.




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


Home page
 Nucleic Acids ResHome page
R. M. Cawthon
Telomere measurement by quantitative PCR
Nucleic Acids Res., May 15, 2002; 30(10): e47 - e47.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 1999 by the American Association for Clinical Chemistry.