Detection of DNA targets with biotinylated and fluoresceinated RNA probes. Effects of the extent of derivitization on detection sensitivity.
The substituted nucleotide aminohexyl-ATP (AH-ATP) was used for synthesis of RNA probes from a plasmid template using the T7 phage promoter. Following synthesis, RNA probes were modified by reaction with N-hydroxysuccinimide (NHS) esters of biotin or fluorescein. Nearest-neighbor analysis was used to quantitate both the incorporation of the substituted nucleotide into RNA and the subsequent modification of the incorporated nucleotide by the NHS esters. The results indicate that AH-ATP is efficiently incorporated into RNA and that modification of the amine group is also efficient. The T7 polymerase shows a bias for ATP over AH-ATP and truncated transcripts are produced if 100% AH-ATP is used for synthesis. However, the use of 50% AH-ATP in the synthesis reaction yields full-length RNA probes that contain on average one amine-labeled nucleotide every 12 bases. This RNA is readily modified by the respective NHS esters to obtain one biotin group per 15-18 total RNA bases or one fluorescein group per 25-35 bases. Probes modified with biotin or fluorescein were used to detect picogram levels of target DNA in a dot blot hybridization format.
Folsom
V
V
Bethesda Research Laboratories, Life Technologies, Inc., Gaithersburg, Maryland 20877.
Hunkeler
M J
MJ
Haces
A
A
Harding
J D
JD
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000032:analysis; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001710:Biotin; D002855:Chromatography, Thin Layer; D004247:DNA / Q000032:analysis*; D005452:Fluoresceins; D006801:Humans; D015151:Immunoblotting; D012313:RNA / Q000032:analysis / Q000096:biosynthesis; D015347:RNA Probes / Q000138:chemical synthesis*; D012685:Sepharose
D005452:Fluoresceins; D015347:RNA Probes; C062509:N(6)-aminohexyladenosine monophosphate; D000249:Adenosine Monophosphate; C062428:N(6)-aminohexyladenosine triphosphate; D012313:RNA; D001710:Biotin; D000255:Adenosine Triphosphate; D004247:DNA; D012685:Sepharose