2′-MODIFIED OLIGONUCLEOTIDES FROM METHOXYOXALAMIDO AND SUCCINIMIDO PRECURSORS: SYNTHESIS,PROPERTIES, AND APPLICATIONS

Synthesis of 2′-modified oligonucleotides from 2′-methoxyoxalamido (MOX) and 2′-succinimido (SUC) precursors is described. Their physical and biochemical properties were assessed. Synthesized oligonucleotides were used as primers in advanced DNA sequencing protocols. An example of sequencing directly off genomic DNA template without prior cloning or PCR amplification is presented.     

Rapid removal of unincorporated label and proteins from DNA sequencing reactions

This article presents a simple and rapid method for removal of unincorporated label and proteins from DNA sequencing reactions by using Wizard purification resin. This method can be successfully applied for preparation of end-labeled oligonucleotides free of unincorporated label, which is important in experiments (including DNA sequencing) when the level of background should be as low as possible. Also, this method is effective in removal of proteins from DNA sequencing reactions.     

DNA sequencing by hybridization to microchip octa-and decanucleotides extended by stacked pentanucleotides.

The efficiency of sequencing by hybridization to an oligonucleotide microchip grows with an increase in the number and in the length of the oligonucleotides; however, such increases raise enormously the complexity of the microchip and decrease the accuracy of hybridization. We have been developing the technique of contiguous stacking hybridization (CSH) to circumvent these shortcomings. Stacking interactions between adjacent bases of two oligonucleotides stabilize their contiguous duplex with DNA. The use of such stacking increases the effective length of microchip oligonucleotides, enhances sequencing accuracy and allows the sequencing of longer DNA. The effects of mismatches, base composition, length and other factors on the stacking are evaluated. Contiguous stacking hybridization of DNA with immobilized 8mers and one or two 5mers labeled with two different fluorescent dyes increases the effective length of sequencing oligonucleotides from 8 to 13 and 18 bases, respectively. The incorporation of all four bases or 5-nitroindole as a universal base into different positions of the 5mers permitted a decrease in the number of additional rounds of hybridization. Contiguous stacking hybridization appears to be a promising approach to significantly increasing the efficiency of sequencing by hybridization.     

Modifications of guanine bases during oligonucleotide synthesis.

Guanine bases are sensitive to modification during automated DNA synthesis and processing reactions. Methods for the detection of two types of guanine modifications are described. The first method uses the higher reactivity of the modified G base to KMn04 oxidation than T bases, and thus allows detection by chemical DNA sequencing. The second method makes use of the Escherichia coli nucleotide excision repair enzyme UvrABC endonuclease which can detect "bulky" base modifications at each nucleotide in the synthetic DNA. Though the chemical structures of the two modifications are not known, they may be related. Both types of G modifications are often found in oligonucleotides synthesized by the methoxy-diisopropyl-phosphoramidite (MEDP) chemistry but non-detectable in the products of the beta-cyanoethyl-diisopropyl-phosphoramidite (CEDP) chemistry. The Rubin and Schmid pyrimidine-specific chemical DNA sequencing procedure (Rubin, C.M., and Schmid, C.W. (1980) Nucleic Acids Res. 8, 4613-4619) was found to be applicable to oligonucleotides synthesized by the CEDP chemistry, and to oligonucleotides synthesized by the MEDP chemistry if precautionary measures are taken to destroy the signals produced by the highly KMnO4 sensitive modified guanine bases. We also show how chemical DNA sequencing might be useful for diagnosing other chemical modifications in synthetic oligonucleotides.     

Solid-phase methods for sequencing of nucleic acids I. Simultaneous sequencing of different oligodeoxyribonucleotides using a new, mechanically stable anion-exchange paper.

A solid-phase method for simultaneous sequencing of large numbers of oligodeoxyribonucleotides has been developed using a new, mechanically stable anion-exchange paper. The excellent mechanical properties of the polymer allow the processing of several paper segments in one reaction vessel or to carry out all necessary operations on a larger area of the paper. In addition, DNA material can be chemically eluted from the new carrier during the piperidine reaction, thus avoiding salt elution of DNA and subsequent ethanol precipitation steps - a prerequisite for sequencing oligonucleotides. The approach involves 7 operations including: i) immobilization; ii) washing; iii) modification; iv) washing; v) sorting of the papers; vi) piperidine reaction and chemical elution and vii) lyophilization. All steps can be carried out in 4 to 5 hours independently of the number of oligonucleotides to be sequenced. It is also possible to sequence small oligonucleotides with 3 to 4 base pairs. The method can be fully automated.     

Identification of apolipoprotein E polymorphism by using synthetic oligonucleotides

A procedure based on selective hybridization with allele-specific oligonucleotides was developed for typing apolipoprotein E variants from human genomic DNAs. Two sets of oligonucleotides were synthesized and used to discriminate either between epsilon 3 and epsilon 4 alleles or between epsilon 3 and epsilon 2 alleles. Combination of the allele-specific oligonucleotide hybridization with the method for in vitro DNA amplification (Polymerase Chain Reaction) Saiki, R. K. et al. 1985. Science, 230: 1350-1354) (1) dramatically improved the sensitivity and the reliability of the procedure. Adaptation of a simple strategy involving direct cloning and DNA sequencing of in vitro amplified DNA enables rapid identification of any mutation within the apoE gene area encoding the receptor binding domain.     

Rational de novo gene synthesis by rapid polymerase chain assembly (PCA) and expression of endothelial protein-C and thrombin receptor genes

The assembly of synthetic oligonucleotides into genes and genomes is an important methodology. Several methodologies for such synthesis have been developed, but they have two drawbacks: (1) the processes are slow and (2) the error frequencies are high (typically 1-3 errors/kb of DNA). Thermal damage is a major contributor to biosynthetic errors. In this paper, we elucidate the advantages of rapid gene synthesis by polymerase chain assembly (PCA) when used in combination with smart error control strategies. We used a high-speed thermocycler (PCRJet) to effectively minimize thermal damage and to perform rapid assembly of synthetic oligonucleotides to construct two different genes: endothelial protein C receptor (EPCR) and endothelial cell thrombin receptor, thrombomodulin (TM). First, the intact EPCR gene (EPCR-1, 612 bp) and a mutant EPCR-2 (576 bp) that lacked 4 N-linked glycosylation sites were constructed from 35 and 33 oligonucleotides, respectively. Next, for direct error comparison, another longer gene, the 1548 bp TM gene was constructed from 87 oligonucleotides by both rapid and conventional PCA. The fidelity and accuracy of the synthetic genes generated in this manner were confirmed by sequencing. The combined steps of PCA and DNA amplification are completed in about 10 and 22 min for EPCR-1, 2 and TM genes, respectively with comparable low errors in the DNA sequence. Furthermore, we subcloned synthetic TM, EPCR-1, EPCR-2 and native EPCR-1 (amplified from cDNA) into a Pichia pastoris expression vector to evaluate the expression ability, and to compare them with the native gene. Here, we illustrate that the synthetic genes, assembled by rapid PCA, successfully directed the expression of functional proteins. And, importantly, the synthetic and the native genes expressed proteins with the same efficiency.     

��Shotgun DNA synthesis�� for the high-throughput construction of large DNA molecules

We developed a highly scalable ‘shotgun’ DNA synthesis technology by utilizing microchip oligonucleotides, shotgun assembly and next-generation sequencing technology. A pool of microchip oligonucleotides targeting a penicillin biosynthetic gene cluster were assembled into numerous random fragments, and tagged with 20 bp degenerate barcode primer pairs. An optimal set of error-free fragments were identified by high-throughput DNA sequencing, selectively amplified using the barcode sequences, and successfully assembled into the target gene cluster.     

Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library

Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA.     

An oligonucleotide hybridization approach to DNA sequencing

We have proposed a DNA sequencing method based on hybridization of a DNA fragment to be sequenced with the complete set of fixed-length oligonucleotides (e.g., 4(8) = 65,536 possible 8-mers) immobilized individually as dots of a 2-D matrix [(1989) Dokl. Akad. Nauk SSSR 303, 1508-1511]. It was shown that the list of hybridizing octanucleotides is sufficient for the computer-assisted reconstruction of the structures for 80% of random-sequence fragments up to 200 bases long, based on the analysis of the octanucleotide overlapping. Here a refinement of the method and some experimental data are presented. We have performed hybridizations with oligonucleotides immobilized on a glass plate, and obtained their dissociation curves down to heptanucleotides. Other approaches, e.g., an additional hybridization of short oligonucleotides which continuously extend duplexes formed between the fragment and immobilized oligonucleotides, should considerably increase either the probability of unambiguous reconstruction, or the length of reconstructed sequences, or decrease the size of immobilized oligonucleotides.     

Incorporation by chemical synthesis and characterization of deoxyribosylformylamine into DNA.

2-deoxyribosylformylamine is a major oxidative DNA damage type which occurs upon the action of ionizing radiation on DNA. The protected 2-deoxyribosylformylamine phosphoramidite was synthesized and used in conjunction with previously reported alkali labile base protected phosphoramidites ('PAC phosphoramidites') for the preparation of oligodeoxyribonucleotides containing this lesion. Final deprotection of the oligonucleotides was performed under mild alkaline conditions to preserve the integrity of the fragile defect. The presence of formylamino deoxyribosyl residue was confirmed by FAB mass spectrometry sequencing. Oligonucleotides bearing deoxyribosyl formylamine were used as templates for studying in vitro replication. They direct the insertion of guanine or induce a deletion opposite the lesion.     

Rapid identification of clones using the same degenerate oligonucleotide mixture for both screening and sequencing

A simple and rapid strategy for distinguishing between positively hybridizing colonies and false positive-hybridization signals is described. The isolation of a specific DNA sequence depends on the ability to distinguish between a clone that contains the correct sequence and a false hybridization-positive or background signal. This procedure utilizes the same oligonucleotide mixture both as a screening probe and as a sequencing primer. The mixture of oligonucleotides is used as a primer to obtain sequence information directly from double-stranded DNA. Conditions for sequencing with oligonucleotides having up to 64-fold degeneracy are described. Since the sequence information obtained is directly adjacent to the site of oligonucleotide:DNA hybridization, it is necessary to know only a minimal length of DNA or peptide sequence to both design oligonucleotide probes and confirm the authenticity of the hybridization positives. The advantages of the degenerate oligonucleotide sequencing method include the rapid, reliable identification of authentic versus false hybridization positives made directly without subcloning into single-stranded M13 phage, without sequencing large regions of DNA, or without synthesizing sequence-specific primers.     

小鼠白细胞介素4基因的化学合成、克隆与表达

利用381A型DNA合成仪,分29个寡聚核苷酸片段化学合成了小鼠IL-4全基因,共442bp。以pUC12质粒作为载体,将所有合成片段分前后两组进行磷酸化、退火、连接和克隆,经过菌落原位杂交、酶切鉴定和质粒DNA序列分析,分别得到了含有小鼠IL-4前后两半基因片段的两种重组质粒,回收前半基因片段,插入到含有后半基因重组质粒的EcoRI和PstI酶切位点之间,成功地得到了含有小鼠IL-4全基因的重组质粒pFR101。将全合成基因插入到质粒pSM53中,得表达质粒pFR105,转化大肠杆菌TAP106,根据IL-4对CTLL细胞的作用,肯定了TAP106(pFR105)细菌中有小鼠IL-4活性蛋白的表达。     

Site-localized mutagenesis directed by phosphotriester analogs of oligonucleotides

17- and 20-mer oligodeoxyribonucleotides and their analogues, containing one to four phosphate groups esterified with ethyl alcohol in different positions of oligonucleotide chain, were synthesized by modified triester method. Ethylated di- and trinucleotide blocks were prepared by transesterification method from chlorophenyl derivatives. The structures of the oligonucleotides were confirmed by Maxam-Gilbert sequencing method. Oligonucleotides were not totally complementary to the N-terminal region of lac Z'gene (coding for N-terminal fragment of beta-galactosidase) of phage M13mpB DNA and induced the formation of the proposed deletion mutant DNA M13mp1 delta T. Phosphotriester analogues were more effective mutagens as compared to phosphodiester oligonucleotides due to their stability to nucleases. The use of E. coli DNA-polymerase I provided the increase in the mutant yields in case of the phosphotriester analogues. The stability of the analogues to 5'----3'----5'-endonuclease action, the specificity of oligonucleotide: DNA binding and the structure of mutant DNA were studied by the Sanger sequencing method.     

Sequencing by hybridization with the generic 6-mer oligonucleotide microarray: an advanced scheme for data processing

DNA sequencing by hybridization was carried out with a microarray of all 4(6) = 4,096 hexadeoxyribonucleotides (the generic microchip). The oligonucleotides immobilized in 100 x 100 x 20-microm polyacrylamide gel pads of the generic microchip were hybridized with fluorescently labeled ssDNA, providing perfect and mismatched duplexes. Melting curves were measured in parallel for all microchip duplexes with a fluorescence microscope equipped with CCD camera. This allowed us to discriminate the perfect duplexes formed by the oligonucleotides, which are complementary to the target DNA. The DNA sequence was reconstructed by overlapping the complementary oligonucleotide probes. We developed a data processing scheme to heighten the discrimination of perfect duplexes from mismatched ones. The procedure was united with a reconstruction of the DNA sequence. The scheme includes the proper definition of a discriminant signal, preprocessing, and the variational principle for the sequence indicator function. The effectiveness of the procedure was confirmed by sequencing, proofreading, and nucleotide polymorphism (mutation) analysis of 13 DNA fragments from 31 to 70 nucleotides long.     

A facile method for the construction of synthetic genes

A simple protocol for rapid assembly of chemically synthesized deoxyoligonucleotides into double stranded DNA is described. Several parameters of a ligation-free method were investigated to allow efficient assembly of a large number of oligonucleotides into double stranded DNA by polymerase chain reaction. Synthesis of a 701 bp DNA was carried out in a single reaction by assembling 28 oligonucleotides designed with partial overlaps at complementary ends. An estimate of error rate was made by sequencing several independent clones of the synthesized DNA     

Non-radioactive automated sequencing of oligonucleotides by chemical degradation

A non-radioactive sequencing of fluorescently labelled oligonucleotides by solid-phase chemical degradation is described. Although non-radioactive methods have been reported for the dideoxy chain termination technique, such a method has not yet been developed for the chemical degradation sequencing of DNA fragments. A 21-mer fluorescein labelled M13 sequencing primer was sequenced in an on-line automated system in about 30 minutes. The fluorescent dye and its bond to the oligonucleotide were stable during the chemical reactions used for the base specific degradations. As the sequence is determined on-line during electrophoresis, reloading and running 10 fragments simultaneously allows us to use one gel for sequencing of about 50 different oligonucleotides.     

A scalable method for multiplex LED-controlled synthesis of DNA in capillaries

As research in synthetic biology and genomic sciences becomes more widespread, the need for diverse oligonucleotide populations has increased. To limit reagent cost, it would be advantageous to obtain high quality populations in minute amounts. Towards that end, synthesis of DNA strands in capillaries utilizing photolabile 3-nitrophenylpropyloxycarbonyl (NPPOC) chemistry and ultraviolet-light emitting diodes (UV-LEDs) was examined. Multiple oligonucleotides were made in single capillaries and were characterized by hybridization, sequencing and gene synthesis. DNA synthesized in capillaries was capable of being hybridized and signal intensities correlated with microarray data. Sequencing demonstrated that the oligonucleotides were of high quality (up to 44% perfect sequences). Oligonucleotides were combined and used successfully for gene synthesis. This system offers a novel, scalable method to synthesize high quality oligonucleotides for biological applications.     

Synthesis of 3'-3'-linked oligonucleotides branched by a pentaerythritol linker and the thermal stabilities of the triplexes with single-stranded DNA or RNA

Synthesis of 3'-3'-linked oligonucleotides branched by a pentaerythritol linker is described. The branched oligonucleotides were synthesized on a DNA/RNA synthesizer using a controlled pore glass (CPG) with a pentaerythritol linker carrying 4,4'-dimethoxytrityl (DMTr) and levulinyl (Lev) groups. The stability of the triplexes between the branched oligonucleotides and the target single-stranded DNA or RNA was studied by thermal denaturation. The oligonucleotides with the pentaerythritol linker formed thermally stable triplexes with the single-stranded DNA and RNA. Furthermore, the branched oligonucleotides containing 2'-O-methylribonucleosides, especially the oligonucleotide composed of 2'-deoxyribonucleosides and 2'-O-methylribonucleosides, stabilized the triplexes with the single-stranded DNA or RNA. Thus, the branched oligonucleotide containing 2'-O-methylribonucleosides may be a candidate for a novel antisense molecule by the triplex formation.