title: Evaluation of new linkers and synthetic methods for internal modified oligonucleotides.
abstract: Several fluorescence resonance energy transfer (FRET) oligonucleotide probes were made with different internal linkages between the DNA and the quencher dye. In one example, a 5'-fluorescein beta-actin-based 26-mer DNA sequence was synthesized bearing an internal Tamra quencher. Two different versions were prepared using either conventional C5 [N-(6-aminohexyl)-3-acrylamido]pyrimidine-modified uridine and solution-phase Tamra active ester coupling or solid-phase addition of a Tamra amidite to a C5 [N-(6-hydroxyhexyl)-3-acrylamido]pyrimidine-modified uridine. The products were compared in functional assays. They performed very similarly both in a fluorescence-based melting point assay as well as in quantitative PCR. Another set of beta-actin probes were synthesized utilizing N4 [N-2-(ethylene glycol ethyl)-5-methyl]cytidine and solid-phase Tamra amidite addition at positions flanking those of the uridine. These versions gave lower T(m)s than either uridine-labeled probe and did not work as well in quantitative PCR. A control experiment using oligonucleotides with the same modified residues but without fluorophores attached revealed the same trend as the T(m) study of internal Tamra-labeled probes. Experimental details for the synthesis, purification, and testing are presented.
authors.affiliation: Biosearch Technologies, Inc., 81 Digital Drive, Novato, California 94949, USA. Troy.Walton@colorado.edu
authors.forename: Troy A
authors.initials: TA
authors.lastname: Walton
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Lyttle
authors.forename: Daren J
authors.initials: DJ
authors.lastname: Dick
authors.forename: Ronald M
authors.initials: RM
authors.lastname: Cook
mesh_terms: D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D015345:Oligonucleotide Probes / Q000138:chemical synthesis* / Q000592:standards; D016133:Polymerase Chain Reaction; D012235:Rhodamines / Q000737:chemistry; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D012235:Rhodamines