Specific termination of RNA polymerase synthesis as a method of RNA and DNA sequencing.

Termination of RNA synthesis with 3'-O-Methylnucleoside 5'-triphosphates have been studied using E. coli RNA polymerase holoenzyme and poly [d(A-T)] as well as unfractionated T7 D delta III DNA as templates. It was shown that the termination can be used for DNA sequencing. A sequence of a part of RNA synthesized from AI promoter of the DNA have been determined. Syntheses of four 3'-O-Methylnucleoside 5'-triphosphates are described.     

DNA and RNA sequencing utilizing phosphorothioate chemistry

A method for nucleic acid sequencing has been developed based on the observation that phosphorothioate diesters are hydrolysed by treatment with 2-iodoethanol in a solution of aqueous ethanol. For DNA sequencing, primed single-stranded M13 DNA is polymerised with the Klenow fragment of DNA polymerase I in the presence of the three normal deoxyribonucleotide triphosphates and one alpha-phosphorothioate derivative. This is followed by treatment with 2-iodoethanol, precipitation of the DNA fragments and analysis by polyacrylamide electrophoresis. RNA transcribed from plasmids containing the SP6 RNA polymerase promoter is sequenced by including the alpha-phosphorothioate derivative of the ribonucleotide triphosphates in the polymerisation and treating the product with iodoethane. The cleavage reaction involves alkylation of the sulfur atom to form the phosphorothioate triester and hydrolysis catalysed by an adjacent hydroxyl group.     

Hybridization methods for DNA sequencing.

I have conducted a general analysis of the practicability of using oligonucleotide hybridization to sequence DNA. Any DNA sequence may be sequenced by hybridization with a complete panel of oligonucleotides. However, sequencing DNA segments over 2 kb long requires an unrealistic number of hybridization reactions. The optimal protocol is to hybridize 7-mer or 8-mer mixed oligonucleotide probes to immobilized DNA fragments 80 bp long: should this prove impractical, hybridization of labeled 270-bp fragments to immobilized mixed 10-mers is a potential alternative. Both protocols require no more experiments to sequence large regions of DNA than conventional m13-based sequencing and are much easier to automate, thus reducing the requirements for skilled personnel. In the ideal case, hybridization sequencing reduces the number of experiments required to sequence megabase DNA by 90%.     

DNA合成技术及应用

DNA从头合成技术是指以寡核苷酸链为起始的合成DNA片段的技术,其不断进步是合成生物学快速发展的基石之一。常规使用的连接介导的DNA合成技术和PCR介导的DNA合成技术日益成熟,精确合成长度已经达到0．5—1kb。微阵列介导的DNA合成技术不断发展,其低成本、高通量的特点吸引了人们的注意;而酵母体内DNA合成技术的成功探索也为体外DNA合成提供了一种补偿方法。DNA合成在优化密码子用于异源表达、构建异源代谢途径、合成人工基因组以及合成减毒病毒用于疫苗研制等方面有广泛应用。综述了DNA从头合成技术的研究进展,并介绍了DNA合成的前沿应用。     

Coupling of polyoma DNA and RNA synthesis

Polyoma virus RNA from infected mouse embryo cells was examined by gel electrophoresis and in nucleic acid hybridisation experiments. The extent of representation of the polyoma genome in RNA sequences when cytosine arabinoside is present throughout infection is 30 to 40% of that at late times in infection. When viral DNA synthesis is inhibited during the period in which it is rising to its maximum, the resulting cytoplasmic RNA resembles ‘early’ RNA both in size and by its behaviour in competition hybridisation experiments.     

DNA sequencing by synthesis with degenerate primers

The degenerate primer-based sequencing Was developed by a synthesis method(DP-SBS)for high-throughput DNA sequencing,in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays.In this method,adifferent set of degenerate primers containing a give nnumber(n)of degenerate nucleotides at the 3'-ends were annealed to the sequenced templates that were immobilized on the solid surface.The nucleotides(n 1)on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides.The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately.The main advanmge of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension.From the present study,it is found that the DP-SBS method is reliable,simple,and cost-effective for laboratory-sequencing a large amount of short DNA fragments.     

DNA/RNA synthesis and labelling.

Reliable methods of machine-aided RNA synthesis have been established to complement those for DNA assembly. Oligonucleotides containing thio-modified backbones and 2'-O-alkyl sugars head the list of many newly available analogues. Biotin, fluorescent agents and many reporter groups can be conveniently introduced into oligonucleotides in multiples by phosphoramidite or H-phosphonate chemistry.     

The synthesis and use of oligodeoxynucleotides in plasmid DNA sequencing

A convenient procedure for the synthesis and purification of oligonucleotides is described. 16-base long primers synthesised by this method were used to investigate DNA sequencing using plasmid DNA as a template. This allowed the further analysis of the E. coli glt A sequence coding for citrate synthase and enabled determination of the 5'-non-coding regulatory region of the aminoglycoside phosphotransferase gene.     

New rapid methods for DNA sequencing based in exonuclease III digestion followed by repair synthesis.

We describe improve enzymatic methods for sequencing method for sequencing DNA. They are based on partial digestion of duplex DNA with exonuclease III to produce DNA molecules with 3' ends shortened to varying lengths, followed by repair synthesis to extend and label the 3' ends. After asymmetrical cleavage of the DNA with a restriction enzyme, the labeled products are separated by gel electrophoresis and the sequence read from the autoradiogram. The entire procedures, beginning with unrestricted DNA and followed through gel electrophoresis, takes only one day for sequencing both strands of the DNA molecule. These methods are especially suitable for sequencing DNA cloned in plasmid vectors, and they greatly extend the usefulness of the dideoxynucleotide chain termination method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74, 5463, 1977). Using these methods we have determined the sequence of a 410 base pair fragment which includes the yeast SUP3 tyrosine tRNA gene.     

DNA and RNA induced enantioselectivity in chemical synthesis

One of the hallmarks of DNA and RNA structures is their elegant chirality. Using these chiral structures to induce enantioselectivity in chemical synthesis is as enticing as it is challenging. In recent years, three general approaches have been developed to achieve this, including chirality transfer by nucleotide templated synthesis, enantioselective catalysis by RNA/DNAzymes and DNA-based asymmetric catalysis. In this article the concepts behind these strategies as well as the important achievements in this field will be discussed.