Thomas Horn

Nucleic acid probes

This unit describes the physicochemical structures of commonly used nucleic acid probes, classified into four groups based on their mode of nucleic acid binding. The unit provides an excellent background for the many protocols employing these dyes and should be a prerequisite for any studies involving nuclear probes.

A novel method for the rapid detection of specific nucleotide sequences in crude biological samples without blotting or radioactivity; application to the analysis of hepatitis B virus in human serum.

The detection of a little as 0.2 pg (60,000 molecules) of hepatitis B viral (HBV) DNA in human serum samples in 4 h has been demonstrated using a solution-hybridization and bead-capture method. An amplification method based on chemically crosslinked oligodeoxyribonucleotides was coupled with a horseradish peroxidase-labeling scheme for the ultimate detection of the analyte. Two sets of HBV complementary synthetic oligodeoxyribonucleotide probes containing one of two types of single-stranded (ss) overhangs were employed. These ss overhangs were used to capture the probe-analyte complex onto a bead and subsequently to label it. Detection was achieved with either a chemiluminescent or colorimetric output substrate for the enzyme. Only in the presence of the virus was label specifically bound to the support. The assay was relatively unaffected by either sample composition or by the presence of heterologous nucleic acids.

A chemical 5'-phosphorylation of oligodeoxyribonucleotides that can be monitored by trityl cation release

Abstract A new phosphoramidite-derived reagent, (2-cyanoethoxy)-2-(2′-0-4,4′- dimethoxytrityloxyethylsulfonyl)ethoxy-N,N-diisopropylaminophosphine, for the 5′- phosphorylatlon of ollgodeoxyribonucleotides has been developed. Phosphorylatlon efficiency can be determined by the release of 4,4′-dimethoxytrityl cation in acid.

OLIGONUCLEOTIDES WITH ALTERNATING ANIONIC AND CATIONIC PHOSPHORAMIDATE LINKAGES : SYNTHESIS AND HYBRIDIZATION OF STEREO-UNIFORM ISOMERS

Abstract An oligonucleotide containing alternating anionic and stereo-uniform cationic dimethylaminopropyl-phosphoramidate linkages, d(T+T−) 7 T, is shown to bind with unusually high affinity to DNA [poly(dA) and d(C 2 A 15 C 2 )] and RNA [poly(rA)] targets in solutions of low ionic strength. The isomeric oligomer with the opposite configuration at the phosphoramidate links binds much less efficiently.

Hybridization properties of the 5-methyl-isocytidine/isoguanosine base pair in synthetic oligodeoxynucleotides

Abstract The hybridization properties of the 5-methyl-isocytidine/isoguanosine base pair in synthetic oligodeoxynucleotides have been investigated. The 5-methyl-isocytidine/is oguanosine base pair has been determined to be isoenergetic with the cytidine/guanosine, and each base can effectively discriminate mismatches. The synthesis of oligodeoxynucleotides containing 5-methyl-2′-deoxyisocytidine was improved using N 2 -((di-n-butylamino)methylene) protection.

Solid-supported synthesis, deprotection and enzymatic purification of oligodeoxyribonucleotides

Abstract A simple solid-phase scheme for deprotection and enzymatic purification of oligodeoxyribonucleotides synthesized by a phosphoramidite method using hydrazine-sensitive protection is presented.

The Synthesis of Branched Oligonucleotides as Signal Amplification Multimers for Use in Nucleic Acid Assays

Abstract Branched oligonucleotides have been synthesized using phosphoramidite derivatives with two protected hydroxyl functions. These molecules are employed for a label amplification strategy used in DNA probe diagnostics.

Solid Supported Chemical 5′-Phosphorylation of Oligodeoxy-Ribonucleotides that can be Monitored by Trityl Cation Releasi Application to Gene Synthesis

Abstract The gene for human lysozyme was assembled from oligonucleotides that were chemically phosphorylated with a novel phosphorylation reagent.

Controlled Chemical Cleavage of Synthetic DNA at Specific Sites

Abstract In this communication we report the synthesis of a protected abasic molecule, 1′-O-(2-nitrobenzyl)-2′-deoxyriboside, and a special N-4-(6-hydroxyhexyl)-ribocytidine derivative as light- and periodate-sensitive selectable cleavage moieties, respectively, and their use in the characterization of linear and branched single-stranded DNA molecules.

Construction of branched DNA (bDNA) molecules by chemical ligation

Abstract Chemical ligation has been investigated in the assembly of branched DNA (bDNA) molecules. Water soluble coupling reagents were used to ligate single-stranded linear oligomers to bDNA molecules containing 5 and 15 branches. The ligations were particularly efficient when one of the oligomers was partially double-stranded constructed by branching to create a terminal ligation linker (AutoLink). Branched DNA molecules containing more than 1000 nucleotides were assembled.