Synthesis of 5'-oligonucleotide hydrazide derivatives and their use in preparation of enzyme-nucleic acid hybridization probes.
A hydrazone-based method for conjugating synthetic nucleic acids and reporter molecules for use as nonradioactive hybridization probes is presented. Oligonucleotides complementary to the hepatitis B virus were derivatized at their 5' ends with hydrazine or homobifunctional acyl hydrazides. These derivatives reacted facilely with aldehydes to give hydrazones, which were characterized by uv spectroscopy and HPLC. Coupling of aldehyde-modified alkaline phosphatase with carbohydrazide-oligonucleotide derivatives provided a mixture of two enzyme-nucleic acid conjugates in 80-85% yield. The conjugates had a 1:1 and a 2:1 oligonucleotide/enzyme ratio, respectively, and were separated by ion-exchange chromatography. Both conjugates were able to detect 7 amol of target DNA in 1 h, using a colorimetric assay. In contrast, oligonucleotide-horseradish peroxidase conjugates were 40-fold lower in sensitivity of detection.
Ghosh
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SISKA Diagnostics, Inc., La Jolla, California.
Kao
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PM
Kwoh
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DY
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004800:Enzymes, Immobilized / Q000032:analysis; D006834:Hydrazines / Q000378:metabolism*; D008297:Male; D015345:Oligonucleotide Probes / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D013056:Spectrophotometry, Ultraviolet
D004800:Enzymes, Immobilized; D006834:Hydrazines; D015345:Oligonucleotide Probes; D000469:Alkaline Phosphatase