Experimental analysis of gene assembly with TopDown one-step real-time gene synthesis

Herein we present a simple, cost-effective TopDown (TD) gene synthesis method that eliminates the interference between the polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. The method involves two key steps: (i) design of outer primers and assembly oligonucleotide set with a melting temperature difference of >10°C and (ii) utilization of annealing temperatures to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. In addition, we have combined the proposed method with real-time PCR to analyze the step-wise efficiency and the kinetics of the gene synthesis process. Gel electrophoresis results are compared with real-time fluorescence signals to investigate the effects of oligonucleotide concentration, outer primer concentration, stringency of annealing temperature, and number of PCR cycles. Analysis of the experimental results has led to insights into the gene synthesis process. We further discuss the conditions for preventing the formation of spurious DNA products. The TD real-time gene synthesis method provides a simple and efficient method for assembling fairly long DNA sequence, and aids in optimizing gene synthesis conditions. To our knowledge, this is the first report that utilizes real-time PCR for gene synthesis.     

应用PCR-酶切连接法合成全长sFat1基因

人工合成基因在生命科学研究中有着重要的意义, 因此基因合成是一项常用技术。长片段基因的合成比较困难, 常常因为合成中碱基序列的错配、突变等原因而导致失败。研究者们所熟知的几种现行的方法仍然难以解决该问题。本研究在作者自身的工作经验中建立了一种新的基因合成方法, 即PCR-酶切连接法。应用该方法成功地将化学合成的27个寡聚核苷酸片段(每个片段长60～68 bp)拼接组装起来, 获得了完整的总长为1 226 bp的基因sFat-1。整个过程仅采用3轮PCR(共7个反应)、2轮的酶切连接(3个反应), 而且未曾出现任何偏离预期基因序列的差错。该方法步骤较少, 技术简单, 出错极少, 是合成长基因序列很好的选择。     

Psoralen plus near-ultraviolet light: a possible new method for measuring DNA repair synthesis

A new method is proposed to inhibit semiconservative DNA synthesis in cultured cells while DNA repair synthesis is being measured. The cells are treated with the DNA-crosslinking agent Trioxalen (4,5,8-trimethylpsoralen) plus near-ultraviolet light, and consequently 99.5% inhibition of replicative DNA synthesis is achieved. Additional DNA-damaging agents induce thymidine incorporation into the double-stranded regions of the DNA. The new method gave results very similar to those obtained with the benzoylated naphthoylated DEAE (BND) cellulose method using three human fibroblast strains, of which one had deficient capacity for DNA repair synthesis following treatment with gamma rays and methyl methanesulfonate. The advantages of the new method are simplicity and rapidity, as well as the high extent to which replicative DNA synthesis is inhibited.     

A post-processing method for optimizing synthesis strategy for oligonucleotide microarrays

The broad applicability of gene expression profiling to genomic analyses has generated huge demand for mass production of microarrays and hence for improving the cost effectiveness of microarray fabrication. We developed a post-processing method for deriving a good synthesis strategy. In this paper, we assessed all the known efficient methods and our post-processing method for reducing the number of synthesis cycles for manufacturing a DNA-chip of a given set of oligos. Our experimental results on both simulated and 52 real datasets show that no single method consistently gives the best synthesis strategy, and post-processing an existing strategy is necessary as it often reduces the number of synthesis cycles further.     

New effective method for the synthesis of oligonucleotides via phosphotriester intermediates.

A rapid and convenient method for the synthesis of deoxyribooligonucleotides has been developed using the phosphotriester approach. The advantage of this methodology for work in solution was successfully demonstrated in synthesis of a number of DNA fragments up to 32-long. Adaptation of the presented method to solid-phase synthesis allows a pentadecamer to be assembled in 4-5 hours using dinucleotides as coupling units.     

Combinatorial solid phase peptide synthesis and bioassays

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.     

Chemical Synthesis of 80-mer Thymidylic Acid

Chemical synthesis of long-chain oligodeoxyribonucleotides was studied by the method of block condensation in a liquid phase. A method for elongating the chain and the synthesis of oligothymidylic acids up to 80-mer are described.     

A novel convergent method for the synthesis of α-acyl-γ-hydroxylactams and its application in the total synthesis of PI-090 and 091

A novel convergent method for the synthesis of α-acyl-γ-hydroxylactams utilizing the aldol reaction of N-Boc-protected γ-methoxylactams was developed. As the first application of this method for the synthesis of biologically active natural products, the total synthesis of platelet aggregation inhibitors PI-090 and PI-091 were also investigated and successfully achieved.     

Synthesis of oligonucleotides with sequences identical with or analogous to the 3''-end of 16S ribosomal RNA of Escherichia coli: preparation of A-C-C-U-C-C via the modified phosphotriester method.

A combination of two different methods for the synthesis of oligoribonucleotides, i.e. the two-step phosphotriester method with 2-chlorophenyl phosphate as bifunctional phosphate source and the modified triester method with 2,2,2-trichloroethyl 2-chlorophenyl phosphorochloridate as monofunctional phosphate source, is applied for the synthesis of the fully-protected hexaribonucleotide A-C-C-U-C-C. The two-step method is used for the synthesis of the required dinucleotide monophosphates 9, 10 and 11. Application of the modified triester method for the coupling of the oligonucleotide blocks results in the formation of the fully-protected hexamer 15. Furthermore, attention is paid to 2,4,6-triisopropylbenzenesulphonyl 4-nitroimidazolide as a new condensing agent for the coupling of larger oligonucleotide blocks.     

Microwave assisted high yielding preparation of N-protected 2'-deoxyribonucleosides useful for oligonucleotide synthesis

A rapid and high yielding method for the synthesis of precursors of synthons for DNA synthesis, N-protected 2'-deoxyribonucleosides is described, which occur under mild conditions using microwave irradiation. The desired material, N-protected nucleosides, was obtained in 93-96% yield in few minutes. The final products were then characterized by 1H-NMR and MALDI-TOF and compared with the standard samples. The method is amenable to small to moderate scale of synthesis.     

一种多肽固相合成方法与纯化策略研究

目的：多肽与小分子化学药物相比,具有生物活性高、特异性强、不容易产生耐药性等特点,是目前新型药物研发的重点领域。多肽的合成直接影响到多肽药物的作用机制以及药物效果,因此需要建立一种更加便捷、高效的多肽合成方法。方法：采用Fmoc固相合成法合成多肽HF01,通过比较氨基酸连接的反应体系以及氨基酸脱保护的反应体系,从中确定最优体系。利用乙酰化基团进行肽链末端保护,经肽链剪切制备干燥的粗肽,最后采用高效液相色谱仪与高分辨质谱仪联用对粗肽进行纯化。结果：确定多肽合成的连接和脱保护反应体系,并获得纯度高达98.3%的线性多肽。结论：建立了一种高效、便捷的多肽合成及纯化方法,提高了实验室合成多肽的效率,为多肽类药物的研发提供技术支撑。     

Simple and an efficient method for the synthesis of 1-[2-dimethylamino-1-(4-methoxy-phenyl)-ethyl]-cyclohexanol hydrochloride: (+/-) venlafaxine racemic mixtures

A novel synthetic method was developed for the synthesis of venlafaxine using inexpensive reagents. An improvement in the method, in the yield was achieved for the conversion of the venlafaxine. This is an improved version, simple and efficient method for the large-scale synthesis of venlafaxine.     

Programmable reactivity-based one-pot oligosaccharide synthesis

A detailed protocol is described for the application of a programmable one-pot oligosaccharide synthesis methodology to the synthesis of fucosyl GM1. This serves as a general example of the application of this method to the synthesis of any desired oligosaccharide. The method relies on a large database of relative reactivities for differentially protected tolyl thioglycoside donor molecules and a computer program to suggest the best order of addition for assembly of the oligosaccharide in optimal yield and with the fewest operations. The product is a protected form of the desired oligosaccharide isolated in 47% yield, which is then deprotected using standard procedures to provide fucosyl GM1 oligosaccharide (1) in 44% yield. The total time for synthesis of 1 from building blocks 3, 4 and 5 is approximately 4 d, whereas synthesis of the same compound by traditional stepwise procedures would take significantly longer. Protocols for the synthesis of thioglycoside building blocks 3 and 4 are also described.     

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。     

A facile route to paclitaxel C-10 carbamates

A general protocol for the synthesis of paclitaxel C-10 carbamates is described. The method entails MeI-mediated activation of 2'-O-TBS-7-O-TES-10-O-deacetyl-paclitaxel-10-O-carbonylimidazole prior to reaction with amines. This method is effective for the synthesis of paclitaxel C-10 derivatives, including bifunctional molecules.     

A theory for the estimation of DNA synthesis rates by flow cytometry

A method is presented for estimating the rate of DNA synthesis of a cell population by examining the DNA histogram generated by flow cytometry (FCM). The model is based on the use renewal equations to estimate the steady-state fraction of cells in each DNA compartment. The fraction of cells in each compartment is shown to be related to the Laplace transform of the transit time through that compartment. Two methods are introduced for estimating the rate of DNA synthesis utilizing different transit time distributions. One method is shown to be a simplification of the method of Dean and Anderson. The other method allows for variability in the DNA synthesis rate. The effects of quiescent cells are considered and attention is paid to the various assumptions underlying the estimation.     

A simple,universal, efficient PCR-based gene synthesis method: Sequential OE-PCR gene synthesis

Herein we present a simple, universal, efficient gene synthesis method based on sequential overlap extension polymerase chain reactions (OE-PCRs). This method involves four key steps: (i) the design of paired complementary 54-mer oligonucleotides with 18 bp overlaps, (ii) the utilisation of sequential OE-PCR to synthesise full-length genes, (iii) the cloning and sequencing of four positive T-clones of the synthesised genes and (iv) the resynthesis of target genes by OE-PCR with correct templates. Mispriming and secondary structure were found to be the principal obstacles preventing successful gene synthesis and were easily identified and solved in this method. Compensating for the disadvantages of being laborious and time-consuming, this method has many attractive advantages, such as the ability to guarantee successful gene synthesis in most cases and good allowance for Taq polymerase, oligonucleotides, PCR conditions and a high error rate. Thus, this method provides an alternative tool for individual gene synthesis without strict needs of the high-specialised experience.     

Isolation of Tyr- mutants of facultative methylotrophic Pseudomonas sp. M

A method for isolation of Tyr- mutants of facultative methylotrophic bacteria Pseudomonas sp. M which possess two tyrosine synthesis pathways is presented. The method is based on the two-step blocking of the tyrosine synthesis: the first step of the supplementary pathway of synthesis from phenylalanine, the second being the main pathway from 4-hydroxyphenylpyruvate.