Preparation of enzyme-conjugated DNA probe and application to the universal probe system.
A general procedure for the cross-linking of enzyme to DNA has been developed for use as a nonradioactive probe. In this method, DNA is transaminated with diaminopropane to introduce primary amino groups into the cytosine residues. Then the amino groups are converted to thiol groups using a heterobifunctional cross-linker. The thiolated DNA is conjugated with the maleimide-introduced enzyme. With this method, alkaline phosphatase was cross-linked to a single-stranded DNA (sspUCRf1). The conjugate was able to detect 5 pg of target DNA (pUCf1 plasmid, 3.2 kbp) fixed onto the nitrocellulose membrane, using a colorimetric assay. The enzyme-conjugated DNA was applied to "the universal probe system," which consisted of two single-stranded DNA probes (a primary probe and a labeled secondary probe). Using alkaline phosphatase-conjugated sspUCRf1 DNA as the secondary probe, the c-myc gene and HBV DNA were detected effectively on Southern and dot-blot hybridization.
Nakagami
S
S
Institute for Biotechnology Research, Wakunaga Pharmaceutical Co., Ltd., Hiroshima, Japan.
Matsunaga
H
H
Oka
N
N
Yamane
A
A
D000469:Alkaline Phosphatase / Q000737:chemistry*; D000586:Amination; D015139:Blotting, Southern / Q000379:methods*; D003124:Colorimetry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D015342:DNA Probes / Q000737:chemistry*; D004229:Dithiothreitol; D006801:Humans; D008301:Maleimides; D009693:Nucleic Acid Hybridization; D013388:Succinimides
D003432:Cross-Linking Reagents; D015342:DNA Probes; D008301:Maleimides; D013388:Succinimides; C070251:N-((4-(2-maleimidoethoxy)succinyl)oxy)succinimide; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D000469:Alkaline Phosphatase; D004229:Dithiothreitol