Brugia malayi and Brugia pahangi: synthetic biotin labeling of oligonucleotide probes for use in species-specific detection assays.
We have developed a novel, high-yield synthetic approach for the incorporation of multiple biotin residues into a series of species-specific oligonucleotide probes for the detection of filarial parasites. The probes are designed to detect species-specific regions of a highly repeated DNA sequence (HhaI repeat) found in all species of Brugia. The synthetic method described in this paper was used to construct oligomer probes tailed on the 5' end with 1 to 46 biotinylated uridine residues. Probes with 46 biotins were found to be more sensitive than probes with 30 or fewer biotins. We also found that alternating the biotinylated uridine residues with nonbiotinylated thymidine residues improved the sensitivity of the probes. Melting temperature studies indicated that the long tails (up to 91 nucleotides) had only a minimal effect on the Tm of the probes. Conditions were found that optimized the sensitivity of the probes while maintaining their species specificity. Using these conditions, the probes were shown to be sensitive enough to detect single parasites in blood using a chemiluminescent detection system. This method of nonradioactively labeling oligonucleotides for the detection of infectious agents will enable the use of such probes in endemic regions in developing countries.
Department of Biological Sciences, Edna McConnell Clark Science Center, Smith College, Northampton, Massachusetts 01063.
S A
SA
Williams
C B
CB
Poole
D
D
Landry
J
J
Glover
L A
LA
McReynolds
D000818:Animals; D001483:Base Sequence; D001710:Biotin; D017178:Brugia malayi / Q000235:genetics / Q000302:isolation & purification*; D017179:Brugia pahangi / Q000235:genetics / Q000302:isolation & purification*; D004247:DNA / Q000235:genetics; D008842:Microfilariae / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D012680:Sensitivity and Specificity; D013045:Species Specificity
D015345:Oligonucleotide Probes; D001710:Biotin; D004247:DNA