Electrophoretic quantitation of nucleic acids without amplification by single-molecule imaging

Takashi Anazawa, Hiroko Matsunaga, Edward S. Yeung*

*この研究の対応する著者

研究成果: Article査読

48 被引用数 (Scopus)

抄録

We have developed a novel high-performance quantitative assay for unamplified nucleic acids that is based on single-molecule imaging. The apparatus is a simple but highly sensitive single-molecule detection system that uses a normal CCD camera instead of an image-intensified CCD camera. After the DNA molecules in a sample were labeled with YOYO-1, they were induced to migrate electrophoretically in a polymer solution and imaged. No chemical or biochemical amplification was required. Direct quantitation of the sample by counting molecules was possible, because the number counted over the measurement period was directly proportional to the concentration of DNA molecules in the sample. Nonspecifically labeled impurities that would degrade the sensitivity of the assay were successfully reduced and discriminated from the DNA molecules by differences in electrophoretic mobility. By using β-actin DNA (838 bp) as a model sample, we demonstrate that this protocol was fast (10-min measurement period), highly sensitive (limit of quantitation: ∼103 copies/sample, or 3 × 10-16 M), quantitative, and covered a wide linear dynamic range (∼104). This high-performance assay promises to be a powerful technology for the quantitation of specific varieties of mRNA in the study of gene functions and diseases and in the clinical detection of mutant cells.

本文言語English
ページ(範囲)5033-5038
ページ数6
ジャーナルAnalytical chemistry
74
19
DOI
出版ステータスPublished - 2002 10 1
外部発表はい

ASJC Scopus subject areas

  • 分析化学

フィンガープリント

「Electrophoretic quantitation of nucleic acids without amplification by single-molecule imaging」の研究トピックを掘り下げます。これらがまとまってユニークなフィンガープリントを構成します。

引用スタイル