| Budget Amount *help |
¥105,040,000 (Direct Cost: ¥80,800,000、Indirect Cost: ¥24,240,000)
Fiscal Year 2006: ¥9,750,000 (Direct Cost: ¥7,500,000、Indirect Cost: ¥2,250,000)
Fiscal Year 2005: ¥9,750,000 (Direct Cost: ¥7,500,000、Indirect Cost: ¥2,250,000)
Fiscal Year 2004: ¥18,070,000 (Direct Cost: ¥13,900,000、Indirect Cost: ¥4,170,000)
Fiscal Year 2003: ¥23,920,000 (Direct Cost: ¥18,400,000、Indirect Cost: ¥5,520,000)
Fiscal Year 2002: ¥43,550,000 (Direct Cost: ¥33,500,000、Indirect Cost: ¥10,050,000)
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| Research Abstract |
Chemiluminescence-imaging technology has been widely used in many fields of research, and a more facile and sensitive DNA-detection method has been desired for microanalysis. We studied novel detection methods of DNA utilizing non-enzymatic chemiluminescent macromolecular probes, in which many luminol and biotin molecules are conjugated into one dextran molecule, or trimethoxyphenylglyoxal (TMPG)-labeled chemiluminescent guanines and biotin are conjugated into large DNA. These probes can be tethered with avidin to form a probe-chained assembly based on the interaction between free avidins and biotins in the probe.
Several chemiluminescent probes having luminol or isoluminol and biotin were synthesized using different sizes of dextran. The chemiluminescence intensity of the probes was increased with increased number of luminol incorporated into the dextran. DextranT2000 (average MW, 2,000 kDa) containing 2627 molecules of luminol and 385 molecules of biotin was used for the detection of
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telomere DNA. This probe afforded the sensitive detection of the target DNA at sub-picomole level, while problematic background signals were observed in a comparative study using a widely used horseradish peroxidase. The current method permitted a promising chemiluminescence-imaging detection of the target DNA.
On the other hand, we utilized a unique chemical derivatization reagent, TMPG. This reagent reacted specifically with guanine bases in nucleic acids to produce quickly chemiluminescent derivatives under mild reaction conditions. TMPG gave an increasing CL intensity depending on the content of guanine base in the DNA molecule at picomole level. Thus we tried immobilized-hybridization assay of the telomere DNA binding to its cDNA on a nylon membrane.
The TMPG detection with CCD camera allowed the high sensitivity as low as femtomole level of the telomere DNA after binding to biotinylated large DNA for signal amplification by the reaction between avidin and biotin. We are required to reconfirm the biotylation of large DNAs. Less
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