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 Improved Method of Gene Synthesis Based on DNA Works Software and Overlap Extension PCR

A bottleneck in recent gene synthesis technologies is the high cost of oligonucleotide synthesis and post-synthesis sequencing. In this article, a simple and rapid method for low-cost gene synthesis technology was developed based on DNAWorks program and an improved single-step overlap extension PCR (OE-PCR). This method enables any DNA sequence to be synthesized with few errors, then any mutated sites could be corrected by site-specific mutagenesis technology or PCR amplification-assembly method, which can amplify different DNA fragments of target gene followed by assembly into an entire gene through their overlapped region. Eventually, full-length DNA sequence without error was obtained via this novel method. Our method is simple, rapid and low-cost, and also easily amenable to automation based on a DNAWorks design program and defined set of OE-PCR reaction conditions suitable for different genes. Using this method, several genes including Manganese peroxidase gene (Mnp) of Phanerochaete chrysosporium (P. chrysosporium), Laccase gene (Lac) of Trametes versicolor (T. versicolor) and Cip1 peroxidase gene (cip 1) of Coprinus cinereus (C. cinereus) with sizes ranging from 1.0 kb to 1.5 kb have been synthesized successfully. Bingxue Dong and Runqian Mao contributed equally to this work.     

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1α) promoter to drive gene expression. Plasmid vectors containing hEF1α are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1α promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1α promoter part 1, hEF1α promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.     

Effect of salicylic acid,methyl jasmonate,ethephon and cantharidin on anthraquinone production by Rubia cordifolia callus cultures transformed with the rolB and rolC genes

A novel method for immobilizing large DNA fragments on a solid surface was developed. A mixed self-assembled monolayer of thiolated single-stranded DNA with inert alkanethiol was generated on a gold (Au) surface through the Au-S reaction. Surface-tethered DNA generated by this method was compatible with various genetic engineering techniques, including hybridization, polymerization, restriction enzyme digestion and ligation. Kinetic control of surface coverage of immobilized DNA was critical for optimizing genetic engineering techniques on solid-phase. Multi-step reaction schemes utilizing various genetic engineering techniques described above were employed for solid-phase gene assembly. We were able to immobilize DNA fragments of up to 1180 bp on a solid surface. Furthermore, we showed that these immobilized genes can be regenerated by PCR. The present work suggests that these types of assembled genes can be used to store and regenerate genes on solid-phase.     

Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis

A basic problem in gene synthesis is the acquisition of many short oligonucleotide sequences needed for the assembly of genes. Photolithographic methods for the massively parallel synthesis of high-density oligonucleotide arrays provides a potential source, once appropriate methods have been devised for their elution in forms suitable for enzyme-catalyzed assembly. Here, we describe a method based on the photolithographic synthesis of long (>60mers) single-stranded oligonucleotides, using a modified maskless array synthesizer. Once the covalent bond between the DNA and the glass surface is cleaved, the full-length oligonucleotides are selected and amplified using PCR. After cleavage of flanking primer sites, a population of unique, internal 40mer dsDNA sequences are released and are ready for use in biological applications. Subsequent gene assembly experiments using this DNA pool were performed and were successful in creating longer DNA fragments. This is the first report demonstrating the use of eluted chip oligonucleotides in biological applications such as PCR and assembly PCR.     

DNA interference: a simple and efficient gene-silencing system for high-throughput functional analysis in the fern adiantum

RNA interference (RNAi) has become a powerful tool for determining gene function and is used in a wide variety of organisms. Since it is necessary to generate double-stranded RNA (dsRNA) as an inducer for RNAi, preparation of RNAi-inducing constructs is somewhat cumbersome and time consuming, especially for the thousands of genes used in a genome-wide analysis. To overcome these problems, we have developed a more convenient gene-silencing method in the fern Adiantum using double-stranded DNA (dsDNA) as a model system for functional analysis in plants. Delivery of dsDNA fragments homologous to an endogenous gene into gametophytic cells can induce sequence-specific gene silencing. As it only requires dsDNA fragments homologous to a target gene, PCR-amplified fragments are enough to trigger gene silencing. Maximum gene silencing efficiencies of >90% have been achieved for transformed plants. In addition, simultaneous transfer of dsDNA fragments corresponding to multiple genes still has a silencing effect for individual genes. We term this approach 'DNA interference'.     

Regen: program for designing gene assembly

One of the main problems in constructing synthetic genes is the incorrect hybridisation between the oligonucleotides. The problem is resolved if the sequence uniquely defines the position of the oligonucleotide in the assembled gene. This can be accomplished through the wise partition of dsDNA sequence in the fragments. We describe a program for use in designing such gene assembly. For a given DNA sequence and the approximate location of oligonucleotide boundary it generates all sets of protruding ends that share the smallest homology.     

Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler

Polymerase chain assembly (PCA) is a technique used to synthesize genes ranging from a few hundred base pairs to many kilobase pairs in length. In traditional PCA, equimolar concentrations of single stranded DNA oligonucleotides are repeatedly hybridized and extended by a polymerase enzyme into longer dsDNA constructs, with relatively few full-length sequences being assembled. Thus, traditional PCA is followed by a second primer-mediated PCR reaction to amplify the desired full-length sequence to useful, detectable quantities. Integration of assembly and primer-mediated amplification steps into a single reaction using a high-speed thermocycler is shown to produce similar results. For the integrated technique, the effects of oligo concentration, primer concentration, and number of oligonucleotides are explored. The technique is successfully demonstrated for the synthesis of two genes encoding EPCR-1 (653 bp) and pUC19 β-lactamase (929 bp) in under 20 min. However, rapid integrated PCA–PCR was found to be problematic when attempted with the TM-1 gene (1509 bp). Partial oligonucleotide sets of TM-1 could be assembled and amplified simultaneously, indicating that the technique may be limited to a maximum number of oligonucleotides due to competitive annealing and competition for primers.     

Chemical-enzymatic synthesis and cloning of the full-length genome of a DNA-containing plant virus]

The full-length genomic DNA of a small plant virus--coconut foliar decay virus (CFDV)--has been synthesized by a combination of chemical synthesis, ligation and polymerase chain reaction. Three separately cloned DNA fragments of the genomic DNA were sequenced and assembled in the pPCV002 plasmid vector.     

Reconstruction of a hybrid nucleoside antibiotic gene cluster based on scarless modification of large DNA fragments

Genetic modification of large DNA fragments(gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity.In this study,we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction(PCR) targeting combined with Gibson assembly.In this strategy,the biosynthetic genes for peptidyl moieties(HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid(CPOAA)from the polyoxin biosynthetic gene cluster to generate a~40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette.The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic,polynik A,was obtained and verified.This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.     

生物信息学研究新基因LRP15

为了利用生物信息学探索网上克隆基因与预测基因功能的新方法,首先用一段1.8kbDNA片段的人类EST数据库中进行电子杂交并对相互重叠的EST片段组装,通过引物设计进行cDNA末端快速扩增,以高通量基因组序列（HTGS）数据库及SAGE库为基础,进行染色体定位及组织表达分析。通过DAS及RPS－BLAST程序预测其蛋白质结构及功能。结果显示,LRP15 cDNA全长1718bp,含一个780bp的开放读码框架,编码259个氨工酸,该基因定位于染色体3p24,在多种组织中表达。其编码蛋白含有N端富含亮氨酸重复序列及潜在的穿膜序列。研究表明,生物信息学是克隆与预测基因功能的有效方法;LRP15基因可能是一个参与细胞发育调控的新基因。     

几种新型植物基因表达载体的构建方法

利用基因工程技术手段研究基因功能过程中,构建基因表达载体处于转基因植物的主导地位,采用合适的构建方法会使实验效果事半功倍。植物基因表达载体的构建方法除了传统构建法、Gateway技术、三段T-DNA法、一步克隆法等,还有近年来出现的几种新型的载体构建方法:基于竞争性连接原理快速构建小片段基因表达载体;MicroRNA前体PCR置换法适用于构建小分子RNA表达载体;重组融合PCR法特别适用于插入片段中含有较多限制性酶切位点的载体构建;利用In-Fusion试剂盒可以将任何目的片段插入一个线性化载体的某个区域;构建多片段复杂载体可采用不依赖序列和连接的克隆方法(Sequence and ligation-independent cloning,SLIC)法;Gibson等温拼接法;Golden Gate拼接法。本文将在总结分析前人工作的基础上,结合自己工作的体会和经验分析这7种新方法的特点,期望通过这几种新的方法给植物基因工程表达载体的构建提供新的思路。     

Identification,sequence analysis and expression studies of novel anther-specific genes of Arabidopsis thaliana

Relatively little is known about pollen development at the molecular level. For the purpose of gaining understanding of the molecular control of pollen development, a number of Arabidopsis cDNA fragments were isolated using subtractive hybridizations. DNA and RNA hybridizations and sequence analyses indicate that we have isolated cDNAs representing 13 genes. Sequences for 8 of these genes are novel, while those for the remaining 5 genes have substantial similarity to genes previously reported as anther- or pollen-specific. RNA in situ hybridizations with 5 genes revealed that four of them are tapetum-specific with differing temporal expression patterns during pollen development and one is pollen-specific within the flower. Sequence analysis of full-length cDNAs showed that one of the novel genes, ATA7, encodes a protein related to lipid transfer proteins. Another gene, ATA20, encodes a protein with novel repeat sequences and a glycine-rich domain that shares a predicted structure with a known cell wall protein. The full-length ATA27 cDNA encodes a protein similar to the BGL4 -glucosidase from Brassica napus. The ATA27 protein is predicted to have an ER retention signal and an acidic isoelectric point, suggesting that it may be localized to the ER lumen. This may be a means of compartmentalization from its substrate(s). Our studies demonstrate that subtractive hybridizations can be used to identify previously unknown genes, which should be valuable tools for further study of pollen and anther development and function.     

A new method for the synthesis of a structural gene.

A novel method of synthesizing a structural gene or gene fragment, consisting of the first synthesis of a single-stranded DNA (ssDNA), has been developed. As a preliminary test of this method, four synthetic genes or gene fragments have been synthesized. The first one with 396 base pairs (b.p.) codes for the mature rbcS from wheat, the next two with 370 and 342 b.p. respectively, for two half molecules of a gene for trichosanthin and the last one with 315 b.p. for the N-terminal 1-102 residues of human prourokinase. In all these syntheses, a plus-stranded DNA of the target gene was generally assembled by a stepwise or one step T4 DNA ligase reaction of six oligonucleotides (A, *pB, *pC, *pD, *pE and *pF) of 30-71 nucleotides long in the presence of two terminal complementary oligonucleotides (Ab' and eF') and three short inter-fragment complementary oligonucleotides (bc, cd and de). After purification, the synthetic ssDNA was inserted into a cloning vector, pWR13. The resulting product was directly used to transform a host cell. The structure of the cloned synthetic gene was confirmed by DNA sequence analysis.     

A rapid DNA assembling strategy mediated by direct full-length polymerase chain reaction

An efficient DNA assembling strategy was developed here modified from Class-IIS endonuclease mediated DNA splicing by directed ligation (SDL). Benefited from the full-length PCR directly using ligation products as template, this strategy required less effort and less time to obtain the assembled full-length DNA. The advantages of this strategy made it a rapid and easy-to-perform gene splicing and multiple site-directed mutagenesis approach especially practicable when more fragments need to be assembled at the same time.     

Quikgene: a gene synthesis method integrated with ligation-free cloning

Gene synthesis is a convenient tool that is widely used to make genes for a variety of purposes. All current protocols essentially take inside-out approaches to assemble complete genes using DNA oligonucleotides or intermediate fragments. Here we present an efficient method that integrates gene synthesis and cloning into one step. Our method, which is evolved from QuikChange mutagenesis, can modify, extend, or even de novo synthesize relatively large genes. The genes are inserted directly into vectors without ligations or subcloning. We de novo synthesized a 600-bp gene through multiple steps of polymerase chain reaction (PCR) directly into a bacterial expression vector. This outside-in gene synthesis method is called Quikgene. Furthermore, we have defined an overlap region of a minimum of nine nucleotides in insertion primers that is sufficient enough to circularize PCR products for efficient transformation, allowing one to significantly reduce the lengths of primers. Taken together, our protocol greatly extends the current length limit for QuikChange insertion. More importantly, it combines gene synthesis and cloning into one step. It has potential applications for high-throughput structural genomics.     

An improved chemico-enzymatic method of synthesizing long DNA segments

A simple and economy method of the biochemical assembling of long double-stranded DNA segments is described. A single-stranded polydeoxynucleotide 122 bases long representing a fragment of synthetic gene of human beta-interferon was assembled from three synthetic fragments 36 (two) and 50 bases long on four complementary 12-mers as templates. This single-stranded polynucleotide was converted, in the presence of DNA polymerase 1 and a 12-meric primer, in to the full-length double-stranded DNA (the beta-interferon gene segment). It was cloned into an E. coli plasmid vector pBR322 and its sequence confirmed.     

A simple and accurate two-step long DNA sequences synthesis strategy to improve heterologous gene expression in pichia

In vitro gene chemical synthesis is a powerful tool to improve the expression of gene in heterologous system. In this study, a two-step gene synthesis strategy that combines an assembly PCR and an overlap extension PCR (AOE) was developed. In this strategy, the chemically synthesized oligonucleotides were assembled into several 200-500 bp fragments with 20-25 bp overlap at each end by assembly PCR, and then an overlap extension PCR was conducted to assemble all these fragments into a full length DNA sequence. Using this method, we de novo designed and optimized the codon of Rhizopus oryzae lipase gene ROL (810 bp) and Aspergillus niger phytase gene phyA (1404 bp). Compared with the original ROL gene and phyA gene, the codon-optimized genes expressed at a significantly higher level in yeasts after methanol induction. We believe this AOE method to be of special interest as it is simple, accurate and has no limitation with respect to the size of the gene to be synthesized. Combined with de novo design, this method allows the rapid synthesis of a gene optimized for expression in the system of choice and production of sufficient biological material for molecular characterization and biotechnological application.