Preparation of europium-labelled DNA probes and their properties.
A chemical method for labelling DNA with a europium chelate is presented. First, primary aliphatic amino groups are introduced onto DNA in a transamination reaction. The transamination reaction is altered by adjusting temperature and duration of the reaction. Subsequently, the modified DNA is reacted with an isothiocyanate derivative of a Eu chelate. The optimum amount of Eu chelates on a DNA probe is 4-8% of total nucleotides. There is a decrease of 0.7 degrees C in the melting temperature of DNA for each incorporated Eu chelate on 100 bases. Hybridization efficiency is lowered by the introduction of Eu chelates but this effect can be partly overcome by using high DNA probe concentrations. The detection limit of the Eu-labelled probe is 0.15 attomoles of target DNA in a mixed-phase hybridization assay on microtitration wells. In addition to high sensitivity the Eu-labelled probes offer convenience in use and results which are quantitative and easy to interpret.
Hurskainen
P
P
Dahlén
P
P
Ylikoski
J
J
Kwiatkowski
M
M
Siitari
H
H
Lövgren
T
T
D000345:Affinity Labels; D010582:Bacteriophage lambda / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry* / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D005063:Europium; D008722:Methods; D009693:Nucleic Acid Hybridization; D012680:Sensitivity and Specificity; D013696:Temperature
D000345:Affinity Labels; D015342:DNA Probes; D004279:DNA, Viral; D005063:Europium