Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: I. Optimum conditions and minimum oligodeoxyribonucleotide length. 1979.
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Amino Acid Sequence↗
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Binding of complement by complexes between antibodies to nucleic acid constituents and short oligodeoxyribonucleotides.
Oligodeoxyribonucleotides act as inhibitors of the complement fixation caused by complexes between antibodies to defined oligodeoxyribonucleotides and denatured DNA. At concentrations higher than 50 micrograms oligodeoxyribonucleotide/ml complement fixation occurred in the absence of antigen. The extent of complement binding depends on the specificity of the antibodies as well as on the composition of the oligodeoxyribonucleotides. Complement fixation is observed most strongly with antisera to oligodeoxyribonucleotides and to denatured DNA, which belong predominantly to the IgM class. With two LE-sera, containing antibodies to denatured and to native DNA, no complement fixation was found. It is supposed that specific interactions of the oligodeoxyribonucleotides with amino acid residues closely neighbored to the antibody combining site lead to conformational changes in the antibody molecules and to an activation of the complement binding site.
Animals↗
Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: I. Optimum conditions and minimum ologodeoxyribonucleotide length.
A synthetic oligodeoxyribonucleotide mismatched at a single nucleotide to a specific complementary site on wild-type circular phi X174 DNA can be used to produce a defined point mutation after in vitro incorporation into closed circular duplex DNA by elongation with DNA polymerase and ligation followed by transfection of Escherichia coli (Hutchison et al., 1978; Gillam et al., 1979). The present study is an investigation of the optimum conditions required for the oligodeoxyribonucleotide-primed reaction for production of transition and transversion mutations in phi X174 DNA, using the large (Klenow) fragment of E. coli DNA polymerase I. Under optimum conditions up to 39% of the progeny of transfection are the desired mutant and significant mutation is observed using a heptadeoxyribonucleotide.
Bacteriophage phi X 174↗
Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: II. In vitro selection of mutant DNA.
A method for the in vitro selection of mutant DNA has been devised as an adjunct to the recently developed method for the use of short enzymatically-synthesized oligodeoxyribonucleotides of defined sequence as site-specific mutagens for circular DNA. The selection method uses the mutating oligodeoxyribonucleotide as a primer for Escherichia coli DNA polymerase I (large fragment) under conditions where there is preferential interaction with mutant DNA template. After ligation using T4 DNA ligase, endonuclease S1 is used to degrade single-stranded non-mutant DNA leaving the desired mutant as closed circular duplex DNA. This paper describes the development of the method using mutants in phi X174 DNA as the model system. Studiies on the changes A leads to G and G leads to A at position 587 of phi X174 viral DNA (am3 to wild-type and its reversal) show that one or two cycles of selection can lead to a population of phage consisting of close to 100% mutants.
Bacteriophage phi X 174↗
Co-expression of tyrosine hydroxylase messenger RNA 1 and 2 in human ventral mesencephalon revealed by digoxigenin- and biotin-labelled oligodeoxyribonucleotides.
In situ hybridization experiments, using oligodeoxyribonucleotides specific for the two major expressed human tyrosine hydroxylase mRNAs, were performed on human brain sections at the level of the mesencephalon. The specificity of the probes was ascertained by Northern blot experiments carried out with independently in vitro synthesized human tyrosine hydroxylase mRNAs. For in situ hybridization experiments, oligodeoxyribonucleotides were labelled with nucleotides tagged with digoxigenin or biotin molecules. The hybridized oligonucleotides were detected by antibodies coupled with peroxidase and alkaline phosphatase enzymes, which yield, with appropriate substrates, brown and purple products, respectively. The simultaneous detection of the two mRNAs with digoxigeninated and biotinylated probes revealed that these two mRNAs are co-expressed in single cells. The purple product obtained with alkaline phosphatase exhibits a discrete distribution within the dopaminergic cells suggesting these mRNAs are associated with sub-cellular structures. Finally, a heterogeneity in the intensity of the labelling of reactive cells with both probes was visualized as well as the expression of the two mRNA species in neurites.
Aged↗
T4 RNA Ligase joins 2'-deoxyribonucleoside 3',5'-bisphosphates to oligodeoxyribonucleotides.
T4 RNA ligase catalyzes the ATP-dependent addition of a single 2'-deoxyribonucleoside 3',5'-bisphosphate to the 3'-hydroxyl of an oligodeoxyribonucleotide. The bisphosphate is joined to the deoxyoligomer by a 3' leads to 5' phosphodiester bond and the product, which is terminated by a 3'-phosphate, is one nucleotide longer than the substrate. Bisphosphates of dAdo, dCyd, dGuo, dThd, and dUrd are donors and oligodeoxyribonucleotides with dA, dC, dG, dT, or dU 3' termini act as acceptors. The preferred residue for both donor and acceptor is dCyd. Deoxyoligomers from 3 to 12 residues in length are active as acceptors. To obtain good yields, high concentration of enzyme, long incubation time at low temperature, and manganous rather than Mg(II) ion are required. Under optimal conditions, yields calculated with respect to deoxyoligomer converted to product vary from 40 to greater than 95%. The turnover number of the enzyme for DNA joining is extremely low but, because the preparation is nearly free of DNases, there is less than 3% degradation of substrate or product after 6 days of reaction. We anticipate that this reaction will serve as the basis for a method for the stepwise enzymatic synthesis of DNA of defined sequence.
Adenosine Triphosphate↗
Antibodies elicited by defined oligodeoxyribonucleotide sequences.
Antibodies to the oligodeoxyribonucleotides d(pT)3, d(pT)4, d(pT)6 and d(pA-A-T-T) were elicited in rabbits by immunization with electrostatic complexes of the respective haptens with methylated bovine serum albumin (MBSA). The antisera were assayed by complement fixation using denatured DNA's of various sources as antigens. The specificities of the antibodies were determined by estimating the inhibition of the complement fixation reaction by defined oligodeoxyribonucleotides. The antibodies were shown to be specific for the sequence of the oligode-oxyribonucleotides or parts of it.
Animals↗
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.
Bacteriophage phi X 174↗
Enzymatic synthesis of oligodeoxyribonucleotides of defined sequence.
Procedures for the controlled addition of one or more deoxyribonucleotide residues to the 3' end of an oligodeoxyribonucleotide primer are described. Polynucleotide phosphorylase (EC 2.7.7.8), purified from Escherichia coli B, catalyzes the reaction using a deoxyribonucleoside 5'-diphosphate as substrate, with Mn2+ as cofactor. Reaction occurs rapidly in aqueous solution, and no protecting groups are required, simplifying recovery and purification of the products. The concentrations of sodium chloride and manganous chloride in the incubation mixture are critical to obtaining good yield of the required product. Primers of chain length from 3 to 12 have been extended by up to 9 deoxyribonucleotide residues to obtain oligodeoxyribonucleotides of chain length up to 13. Yields of single addition products varied from 8 to 59%. Factors which influence these yields are discussed. The effects of added polyamines and some organic solvents on the reaction are described. Spermidine or dimethylsulfoxide in the incubation medium tend to favor the addition of several residues of deoxyribonucleotide to the primer.
Base Sequence↗
A simple and efficient method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded plasmid DNA.
A method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded DNA without the necessity for phenotypic selection is described. Plasmids denatured with alkali and purified by adsorption to and elution from nitrocellulose have single-stranded regions where primers can hybridize and serve as templates for a T7 DNA polymerase-catalyzed synthesis of complementary mutant DNA strands. When this procedure was carried out such that the original nonmutant strand contained uracil [method of Kunkel, Proc. Natl. Acad. Sci. USA 82(1985)488-492], mutation frequencies of between 30% and 40% were obtained. The technique has been used to generate mutant genes in plasmids of a wide variety of sizes. The largest plasmid manipulated and successfully mutagenized was 22 kb. The method is rapid and efficient and is not dependent upon either f1 phage vectors or the presence of restriction sites in the vicinity of the sequence targeted for mutation.
Base Sequence↗
Influence of pH on the equilibrium association constants for oligodeoxyribonucleotide-directed triple helix formation at single DNA sites.
The energetics of oligodeoxyribonucleotide-directed triple helix formation for the pyrimidine.purine.pyrimidine structural motif were determined over the pH range 5.8-7.6 at 22 degrees C (100 mM Na+ and 1 mM spermine) using quantitative affinity cleavage titration. The equilibrium binding constants for 5'-TTTTTCTCTCTCTCT-3' (1) and 5'-TTTTTm5CTm5CTm5CTm5CTm5CT-3' (2, m5C is 2'-deoxy-5-methylcytidine) increased by 10- and 20-fold, respectively, from pH 7.6 to 5.8, indicating that the corresponding triple-helical complexes are stabilized by 1.4 and 1.7 kcal.mol-1, respectively, at the lower pH. Replacement of the five cytosine residues in 1 with 5-methylcytosine residues to yield 2 affords a stabilization of the triple helix by 0.1-0.4 kcal.mol-1 over the pH range 5.8-7.6. An analysis of these data in terms of a quantitative model for a general pH-dependent equilibrium transition revealed that pyrimidine oligonucleotides with cytidine and 5-methylcytidine form local triple-helical structures with apparent pKa's of 5.5 (C+GC triplets) and 5.7 (m5C+GC triplets), respectively, and that the oligonucleotides should bind to single sites on large DNA with apparent affinity constants of approximately 10(6) M-1 even above neutral pH.
Animals↗
Sequence analysis of oligodeoxyribonucleotides by mass spectrometry. 2. Application of computerized pattern recognition to sequence determination of di-, tri-, and tetranucleotides.
A novel strategy for the sequence analysis of oligodeoxyribonucleotides has been devised which is based upon the analysis of intact underivatized oligonucleotides by mass spectrometry followed by interpretation of the mass-spectral data by computerized pattern-recognition techniques. The pyrolytic and electron-impact conditions of the mass spectrometer permit the cleavage of oligonucleotides of varying chain length and composition, yielding reproducible fragmentations and characteristic m/e values which can be used to reveal purine and/or pyrimidine base sequence information. The selection of optimum features (which are the ratios of peak heights of specific ions, or the linear combination of such ratios) has been done by an interactive feature selection method employing multidimensional k nearest-neighbor analysis and two-dimensional feature-space plots (nonlinear mappings) of the mass-spectral data. Features have been found which allow 100% classification accuracy in predicting the 5' and 3' terminus of all of the dinucleotides commonly found in DNA. Other specific features have been found which indicate adjacent nucleotides within a tetranucleotide. Knowledge of the adjacent nucleotide pairs present, in conjunction with the information as to the 3' or 5' position of the residues in each pair, permits the reconstruction of the sequence of the tetranucleotide.
Base Sequence↗
Role of apurinic sites in the resistance of methylated oligodeoxyribonucleotides to degradation by spleen exonuclease.
The effect of introducing methyl groups into DNA substrates was studied by using the spleen exonuclease (EC 3.1.4.1), an enzyme which hydrolyses oligonucleotides in a sequential manner by splitting off 3'-phosphomononucleotides starting from the 5'-hydroxyl terminus. Analyses of oligodeoxyribonucleotide 3'-phosphate substrates after reaction in vitro with dimethyl sulphate demonstrated that the resultant methylation pattern differed from the previously found for native DNA, particularly with respect to the relative amounts of 1- and 3-methyladenine produced. Although after treatment with increasing amounts of dimethyl sulphate the substrate became progressively resistant to degradation by the exonuclease, the methylation products themselves were only partially responsible for the observed inhibition of enzyme activity. The incomplete degradation encountered was apparently due to the presence of apurinic sites, which arose as secondary lesions after the spontaneous release of the labile alkyl purines from the methylated substrate. Inhibition of enzyme activity appeared to be competitive, being characterized by constant values for apparent Vmax, and increased values for apparent Km. the interpretation of this, however, is complicated by the complex nature of the substrate, and these aspects are considered in some detail.
Adenine↗
Chemical and enzymatic incorporation of N2-(p-n-butylphenyl)-2'-deoxyguanosine into an oligodeoxyribonucleotide.
An 18mer oligodeoxyribonucleotide containing a N2-(p-n-butylphenyl)-2'-deoxyguanosine (BuPdG) residue at the 3' end has been synthesized by both chemical and enzymatic methods. Chemical synthesis involved attachment of 5'-DMT-BuPdG as the 3'-H-phosphonate to uridine-controlled pore glass (CPG), followed by extension via H-phosphonate chemistry. After oxidation of the backbone, deprotection of bases, and removal from CPG, the uridine residue was removed by periodate cleavage and beta-elimination. The resulting oligomer 3'-phosphate was digested with alkaline phosphatase to give the free BuPdG-18mer. E.coli DNA polymerase I (Klenow) incorporated BuPdGTP at the 3' end of the corresponding 17mer primer annealed to a complementary 29mer template, and the properties of this product were identical to those of chemically synthesized BuPdG-18mer. E.coli DNA polymerase I (Klenow) was unable to extend the BuPdG-18mer, and the 3' to 5' exonuclease activity of the enzyme was unable to remove the modified nucleotide.
Base Sequence↗
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.
Amides↗
Enzymatic synthesis of oligodeoxyribonucleotides of defined sequence. Polynucleotide phosphorylase catalysed synthesis using pyrimidine analog-containing deoxyribonucleoside 5'-diphosphates.
The E. coli polynucleotide phosphorylase-catalysed reaction of the deoxynucleoside 5'-diphosphates of 5-methyldeoxycytidine, N4-hydroxydeoxycytidine, deoxyuridine and 5-mercurideoxyuridine with the primers d(pT-T-A-G) and d(pT-T-T-T-T-T) have been studied under conditions where the primer is extended, predominantly, by one or two nucleotide residues. In experiments with 5-mercurideoxyuridine 5'-diphosphate, no 5-mercurideoxy-uridine-containing oligonucleotides were produced. The other three nucleotide analogs were found to be good substrates for E. coli PNPase and the conditions established for synthesis with these analogs will allow the construction of a number of biologically useful types of oligodeoxyribonucleotide.
Chromatography, High Pressure Liquid↗
Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch.
Oligodeoxyribonucleotides complementary to the DNA of the wild type (wt) bacteriophage phi chi 174 have been synthesized by the phosphotriester method. The oligomers, 11, 14, and 17 bases long, are complementary to the region of the DNA which accounts for the am-3 point mutation. When hybridized to am-3 DNA, the oligonucleotides form duplexes with a single base pair mismatch. The thermal stability of the duplexes formed between wt and am-3 DNAs has been measured. The am-3 DNA:oligomer duplexes dissociate at a temperature about 10 degrees C lower than the corresponding wt DNA:oligomer duplexes. This dramatic decrease in thermal stability due to a single mismatch makes it possible to eliminate the formation of the mismatched duplexes by the appropriate choice of hybridization temperature. These results are discussed with respect to the use of oligonucleotides as probes for the isolation of specific cloned DNA sequences.
Bacteriophage phi X 174↗
Rapid synthesis of oligodeoxyribonucleotides. III. Effect of added carboxylate ion on the efficiency of internucleotide bond formation. Solid-phase synthesis of the dodecanucleotide, d(pT-A-A-C-T-G-C-T-C-A-C-T).
In solid-phase oligodeoxyribonucleotide synthesis the addition of benzoic acid to internucleotide phosphodiester bond-forming reactions lowers coupling yields by far greater amount than anticipated on a simple nucleotide-carboxylate molar ratio. High coupling yields were consistently obtained in synthesis of the dodecanucleotide, d(pT-A-A-C-T-G-C-T-C-A-C-T), using rigorously purified nucleotide starting materials.
Benzoates↗