Simple chemical synthesis and hybridization properties of non-radioactive DNA probes

A simple chemical method for the synthesis of non-radioactive DNA probes is described: triazolyl-containing sequences were built by incorporation of 4-triazolylpyrimidin-2-ones instead of cytidines during oligodeoxyribonucleotide synthesis. The activating triazolyl groups were then displaced by a diamine which was further derivatized by a label, such as biotin. Synthesized DNA probes were oligonucleotides complementary to a cloned human antithrombin III DNA sequence. These probes, containing the same label at different positions of the sequence, were hybridized to their target DNA immobilized on nitrocellulose. Their hybridization specificity and stability were studied. Hybrid detection was performed either colorimetrically by the streptavidin-alkaline phosphatase-based system or by autoradiography after 5'-32P labeling of the probes: 15 fmol (0.05 microgram) of complementary sequence could be visualized in the two cases.     

Efficient methods for attachment of thiol specific probes to the 3''-ends of synthetic oligodeoxyribonucleotides.

Methodology is described for the synthesis of DNA oligomers containing a free 3'-thiol group which can be selectively crosslinked with a wide variety of probes. This chemistry is compatible with both phosphotriester and phosphoramidite solid phase chemistry. Moreover, the sulphydryl group is introduced into the 3'-nucleoside solid support linkage prior to oligonucleotide synthesis. Consequently, no additional coupling steps are required after oligonucleotide synthesis, and isolation of the 3'-thiol oligonucleotide requires only one additional deprotection step. Cross-linking of the thiol-containing oligonucleotide to a fluorescent probe was carried out with high selectivity, in high yield, and under mild conditions.     

Biotin-labeled plasmid DNA probes for detection of epithelium-associated strains of lactobacilli.

Biotin-labeled DNA probes prepared from whole plasmids (5.5 and 4.8 kilobases) of two lactobacillus strains (Lactobacillus delbrueckii 21 and Lactobacillus reuteri 100-23) were used to detect the homologous bacteria in microtome-cryostat-prepared sections of murine forestomach. The forestomach sections were incubated on nylon filter membranes placed on agar plates and, after lysis of the lactobacillus cells and denaturation of their DNA, were used in hybridization experiments with the strain-specific DNA probes. Hybridization of the probes to membranes containing sections from lactobacillus-free mice did not occur. The probes detected the presence of homologous strains of lactobacilli in sections cut from the forestomach of mice harboring one or both of the strains.     

Aryl-beta-C-LNA monomers as universal hybridization probes

High-affinity universal hybridization is demonstrated for oligonucleotides containing the pyrenyl-LNA monomer 6b, 2'-O-Me-RNA monomers and LNA monomers.     

Use of synthetic oligodeoxyribonucleotides and primed cDNA as probes for pea (Pisum sativum L.) RNA and genomic DNA sequences

Two oligonucleotide sequences were synthesised by a solid-phase phosphotriester method. One of these sequences, A was a copy of part of a characterised cDNA clone encoding the basic subunit of legumin, a seed storage protein of Pisum sativum L. (garden pea); the other sequence B was predicted to be complementary to the 5 region of legumin mRNA on the basis of the amino acid sequence of legumin acidic subunits and most likely codon usage. Sequence A was shown to hybridise specifically to a legumin cDNA clone and to legumin mRNA. Sequence B did not hybridise specifically to legumin mRNA and was concluded not to be correctly complementary to legumin mRNA. Sequence A was used as a primer for cDNA synthesis using pea seed mRNA as a template. The cDNA so produced hybridised specifically to a legumin cDNA clone, to legumin mRNA, and to sequences encoding legumin in a restriction digest of pea genomic DNA. It is suggested that such oligonucleotide primed cDNAs may be of general value in probing eukaryotic genomic DNA.     

Rapid synthesis of oligodeoxyribonucleotides. IV. Improved solid phase synthesis of oligodeoxyribonucleotides through phosphotriester intermediates.

A phosphotriester solid phase method on a polyamide support has been used to prepare oligodeoxyribonucleotides up to 12 units long. Compared to solid phase phosphodiester synthesis the new methodology is quicker, more flexible and gives 10-60-fold better overall yields.     

"Run-off" synthesis and application of defined single-stranded DNA hybridization probes.

A simple and efficient method for synthesizing radioactively labeled single-stranded DNA hybridization probes with Thermus aquaticus (Taq) DNA polymerase is described. This is done in a "run-off" polymerization with repeated cycles of denaturation, annealing, and extension. It leads to high yields of a single-stranded DNA of defined length (up to 5000 nt), which is labeled to a high specific activity (1.3 x 10(8) cpm/micrograms DNA). These hybridization probes are equally sensitive as nick-translated DNA probes, but strand specific. This was tested by slot blot hybridization with in vitro-transcribed target RNAs and by Northern blotting. The use of single-stranded DNA hybridization probes combines the benefits of DNA stability and single-strand RNA probes.     

Oligonucleotide dendrimers: synthesis and use as polylabelled DNA probes.

Oligonucleotide dendrimers were synthesized using a novel phosphoramidite synthon, tris-2,2,2-[3-(4,4'-dimethoxytrityloxy) propyloxymethyl]ethyl- N , N -diisopropylaminocyanethoxy phosphoramidite. Label, incorporated using [gamma-32P]ATP and polynucleotide kinase, was increased in proportion to the number of 5'-ends. There was a similar increase in signal when these multiply labelled oligonucleotides were used as probes to oligonucleotide arrays. A dendrimeric oligonucleotide was used successfully as a primer in the PCR. The strand bearing the dendrimer was resistant to degradation by T7 Gene 6 exonuclease making it easy to convert the double-stranded product of the PCR to a multiply-labelled, single-stranded probe.     

The use of synthetic oligonucleotides as hybridization probes. II. Hybridization of oligonucleotides of mixed sequence to rabbit beta-globin DNA.

Two oligonucleotides 14-bases long were synthesized, one complementary to rabbit beta-globin DNA (R beta G14A) and the other with the same sequence except for a single base change (T for C) (R beta G14B). Hybridization conditions were established such that R beta G14A would hybridize to globin DNA while R beta G14B would not. We also synthesized a mixture of 13-base long oligonucleotides (R beta G13Mix), representing eight of the possible coding sequences for amino acids 15-19 of rabbit beta-globin. One of the eight is complementary to globin DNA. R beta G13Mix was found to hybridize specifically to globin DNA under conditions where oligonucleotides forming single base pair mismatches do not. Furthermore, R beta G13Mix was shown to hybridize specifically to colonies containing a plasmid with a globin DNA insert. These results are discussed with respect to a general procedure for screening recombinant clones for those containing DNA coding for a protein of known amino acid sequence.     

Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes.

Short synthetic oligonucleotides have been covalently cross-linked to alkaline phosphatase using the homobifunctional reagent disuccinimidyl suberate. The oligomers, twenty-one to twenty-six bases in length, are complementary to unique sequences found in herpes simplex virus, hepatitis B virus, Campylobacter jejuni and enterotoxigenic Escherichia coli. Each oligomer contains a single modified base with a 12-atom "linker arm" terminating in a reactive primary amine. Cross-linking through this amine results in oligomer-enzyme conjugates composed of one oligomer per enzyme molecule that have full alkaline phosphatase activity and can hybridize to target DNA fixed to nitrocellulose within 15 minutes. The hybrids are detected directly with a dye precipitation assay at a sensitivity of 10(6) molecules (2 X 10(-18) mol) of target DNA in 4 hours development time. The enzyme has no apparent effect on selectivity or kinetics of oligonucleotide hybridization and the conjugates can be hybridized and melted off in a conventional manner.     

Convenient synthesis of arabinonucleoside containing oligodeoxyribonucleotides.

C2 substituted arabinofuranosyluracil derivatives were synthesized and its incorporations into DNA were easily carried out by using post-synthetic modification.     

Use of pteridine nucleoside analogs as hybridization probes

The pteridine nucleoside analog 3-methyl isoxanthopterin (3-MI) is highly fluorescent, with a quantum yield of 0.88, and it can be synthesized as a phosphoramidite and incorporated into oligonucleotides through a deoxyribose linkage. Within an oligonucleotide, 3-MI is intimately associated with native bases and its fluorescence is variably quenched in a sequence-dependent manner. Bend ing, annealing, binding, digestion or cleavage of fluorophore-containing oligonucleotides can be detected by monitoring changes in fluorescence properties. We developed a single step method for detecting annealing of complementary DNA sequences using 3-MI-containing oligonucleotides as hybridization probes. One of the complementary strands contains the fluorophore as an insertion and when annealing occurs, the fluorophore bulges out from the double strand, resulting in increased fluorescence intensity. We have examined the sequence dependency, optimal strand length and impact of multiple fluorophores per strand in terms of brightness and impact on the annealing process. We describe the application of this technique to the detection of positive PCR products using an HIV-1 detection system. This sequence-dependent hybridization technique can result in fluorescence intensity increases of up to 27-fold. Fluorescence intensity increases are only seen upon specific binding to bulge-generating complements, removing issues of high background from non-specific binding.     

Rapid synthesis of oligodeoxyribonucleotides: a new solid-phase method.

A method is described that makes use of a new polyamide resin for the rapid synthesis of short oligodeoxyribonucleotides. The method is illustrated by the preparation of two heptadeoxyribonucleotides, d(pT6-C) and d(pC-A-G-T-G-A-T) using a phosphodiester approach. A further development involved use of phenyl isocyanate as an in situ drying agent, which obviated the need for solvent co-evaporation prior tothe internucleotidic coupling steps. Improved fractionation of thymidyl oligonucleotides was obtained by use of a new microparticulate, silica-based anion-exchanger.     

Defined transversion mutations at a specific position in DNA using synthetic oligodeoxyribonucleotides as mutagens.

The oligodeoxyribonucleotides, pCCCAGCCTCAA, which is complementary to nucleotides 5274--4284 of bacteriophage phi X174 viral DNA , and pCCCAGCCTAAA, which corresponds to the same sequence with a C leads to A change at the ninth nucleotide, were synthesized enzymatically. The second of these oligonucleotides was used as a primer for E. coli DNA polymerase I, from which the 5'-exonculease has been removed by proteolysis (Klenow enzyme), on wild-type phi X174 viral DNA template. After ligation, this yielded closed circular heteroduplex DNA with a G, A mismatch at nucleotide 5276. Transfection of E. coli spheroplasts with the heteroduplex DNA produced phage mutated at this nucleotide (G leads to T in the viral DNA) with high efficiency (13%). The mutant DNA, which corresponds to the gene B mutant am16, was reverted (T leads to G) by the wild type oligonucleotide with an efficiency of 19%. The nucleotide changes were established by sequence determination of the mutated viral DNA using the enzymatic terminator method. The production of specific transversion mutations, together with a previous demonstration of specific transition mutations (1), established that short enzymatically synthesized oligodeoxyribonucleotides can be used to induce any class of single nucleotide replacement with high efficiency and thus provide a powerful tool for specific genetic manipulations in circular genomes like that of phi X174.     

Forks and combs and DNA: the synthesis of branched oligodeoxyribonucleotides.

Nucleoside phosphoramidite derivatives containing two protected primary hydroxyl functions have been incorporated into synthetic oligonucleotides as 'branching monomers'. With selective deprotection, multiple identical copies of an additional oligonucleotide can be incorporated to form fork- or comb-like structures for use as signal amplification materials in nucleic acid hybridization assays.     

Improved preparation and synthetic uses of 3-deoxy-d-arabino-hexonolactone: an efficient synthesis of Leptosphaerin

Acetylation of d-glucono-1,5-lactone and subsequent treatment with triethylamine gave 2,4,6-tri-O-acetyl-d-erythro-hex-2-enono-1,5-lactone. Hydrogenation of the latter in the presence of palladium on carbon yielded 2,4,6-tri-O-acetyl-3-deoxy-d-arabino-hexono-1,5-lactone (5) in almost quantitative yield calculated from gluconolactone. Catalytic hydrogenation of 5 with platinum on carbon in the presence of triethylamine gave 2,4,6-tri-O-acetyl-3-deoxy-d-arabino-hexopyranose in quantitative yield. Deacetylation of 5 gave 3-deoxy-d-arabino-hexono-1,4-lactone, which was converted into 3-deoxy-5,6-O-isopropylidene-2-O-methanesulfonyl-d-arabino-hexono-1,4-lactone (10). The latter was converted into 2-acetamido-2,3-dideoxy-d-erythro-hex-2-enono-1,4-lactone (Leptosphaerin). When 10 was boiled in water in the presence of acid, it gave a high yield of 2,5-anhydro-3-deoxy-d-ribo-hexonic acid.     

One pot solution synthesis of cyclic oligodeoxyribonucleotides.

Several cyclic oligodeoxynucleotides with different base composition and size have been prepared from 5',3'-unprotected linear precursors, using a bifunctional phosphorylating reagent. The final deprotected oligomers have been characterized by 1H- and 31P-NMR. The present procedure is particularly useful for millimolar scale syntheses.