Analytical Sciences

Detection of cRNA Hybridized on a DNA Chip Using a Tetrakis-Acridinyl Peptide Cassette, Consisting of TAP and Partially Self-complementary Oligonucleotide, d[A₁₈(TA)₅₁]

Katsuhiro KAWAAI,*¹ Yasumitsu KONDOH,*² Takahiko NOJIMA,*³ Kazuoki TADA,*¹ Shigeori TAKENAKA,*⁴ Hideo TASHIRO,*² and Tomoko TASHIRO*¹

*1 Department of Chemistry and Biological Science, School of Science and Engineering, Aoyama Gakuin University, Kanagawa 229-8558, Japan
*2 Probing Technology Laboratory, Institute of Physical and Chemical Research (RIKEN), Saitama 351-0198, Japan
*3 Center for International Research on MicroMechatronics (CIRMM), Institute of Industrial Science, The University of Tokyo, Tokyo 153-8505, Japan
*4 Department of Materials Science, School of Engineering, Kyushu Institute of Technology, Kitakyushu 804-8550, Japan

A tetrakis-acridinyl peptide (TAP) cassette, consisting of a double-stranded region of alternating AT sequence bound to TAP and a single stranded overhanging sequence of continuous dA, was prepared by mixing TAP with d[A₁₈(TA)₅₁]. A TAP cassette could be applied to the fluorometric detection of hybridized DNA on the DNA chip, which was prepared by stamping a 45-meric DNA probe onto a gold-coated plastic chip using a high-precision spotter developed at RIKEN. Spots on the DNA chip were imaged by a CCD camera after hybridization with 65-meric target single-stranded DNAs carrying a continuous dA₂₀ sequence (dA tail) on the DNA chip after treatment with a TAP cassette. Their fluorescence intensity on the DNA chip showed a good linear correlation with the concentration of the target DNAs in the range from 10 pM to 1 nM. Fluorescence of their spots derived from the TAP cassette remaining on the surface of the DNA chip through the dA tail of the hybridized target DNA. Furthermore, the TAP cassette could be successfully applied to the quantitative detection of complementary RNAs (cRNAs) prepared from rat brain with reverse transcription and in vitro transcription.

Full-text PDF