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.     

Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands.

A new method is described for the direct construction of synthetic genes by applying a modified version of the polymerase chain reaction (PCR) to crude oligonucleotide mixtures made by automated solid phase DNA synthesis. Construction of the HIV-1 393 bp rev gene and the 655 bp nef gene by this method is illustrated. The sequences for the entire top and bottom strands of rev were each programmed into an automated DNA synthesizer. Following DNA synthesis, the two crude oligonucleotide solutions were mixed together, specific primers were added, and the target gene was amplified by a modified PCR technique. Although the longer (greater than 200 bases) strands comprise a very small percentage of the total DNA after solid phase synthesis, this method uses PCR to 'find' and amplify such strands to create the target gene. The rev gene constructed by this method was found to contain 4 sequence errors, which were subsequently corrected by site-directed mutagenesis. In order to evaluate the source of sequence errors, several nef genes were made from the top and bottom strand DNA synthesis solutions using independent PCR's. Results suggest that sequence errors arose from both DNA synthesis and PCR. The utility of this method in producing a functional gene is demonstrated by expression of rev in E.coli.     

SPLICE: a technique for generating in vitro spliced coding sequences from genomic DNA

We describe a rapid and cost-effective technique for the in vitro removal of introns and other unwanted regions from genomic DNA to generate a single sequence of continuous coding capacity, where tissues required for RNA extraction and complementary DNA synthesis are unavailable. Based on an overlapping fusion-PCR strategy, we name this procedure SPLICE (for swift PCR for ligating in vitro constructed exons). As proof-of-principle, we used SPLICE successfully to generate a single piece of DNA containing the coding region of a five-exon gene, the short-wavelength-sensitive 1 (SWS1) opsin gene, from genomic DNA extracted from the brown lemur, Eulemur fulvus, in only two short rounds of PCR. Where the genomic structure and sequence is known, this technique may be universally applied to any gene expressed in any organism to generate a practical unit for investigating the function of a particular gene of interest. In this report, we provide a detailed protocol, experimental considerations, and suggestions for troubleshooting.     

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.     

PCR技术对人IL-3 cDNA进行定点突变的研究

本文利用PCR技术对人IL-3cDNA体外进行定点突变,将人IL-3cDNA第3位Met,第116位Lys密码子突变为Val密码子GTT。PCR扩增片段核苷酸序列与引物设计相应的cDNA突变体序列完全一致。结果证实此方法比经典寡聚核苷酸方法简单、迅速、成本低、效率高,也为基因的修饰,蛋白质工程研究提供了简便、稳定的方法。     

New insights into the de novo gene synthesis using the automatic kinetics switch approach

Here we present a simple, highly efficient, universal automatic kinetics switch (AKS) gene synthesis method that enables synthesis of DNA up to 1.6 kbp from 1 nM oligonucleotide with just one polymerase chain reaction (PCR) process. This method eliminates the interference between the PCR assembly and amplification in one-step gene synthesis and simultaneously maximizes the amplification of emerged desired DNA by using a pair of flanked primers. In addition, we describe an analytical model of PCR gene synthesis based on the thermodynamics and kinetics of DNA hybridization. The kinetics difference between standard PCR amplification and one-step PCR gene synthesis is analyzed using this model and is validated using real-time gene synthesis with eight gene segments (318-1656 bp). The effects of oligonucleotide concentration, stringency of annealing temperature, annealing time, extension time, and PCR buffer conditions are examined systematically. Analysis of the experimental results leads to new insights into the gene synthesis process and aids in optimizing gene synthesis conditions. We further extend this method for multiplexing gene assembly with a total DNA length up to 5.74 kbp from 1 nM oligonucleotide.     

A simple method for constructing a tagged protein

We have developed a simple method for preparing a tagged protein by PCR. With this method any protein sequence can be easily tagged. The techniques include three steps of DNA restriction, ligation and PCR. We could obtain a DNA construct containing SUMO-1 gene with His6 tag sequence with high efficiency by the next day.     

PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors

We introduce a PCR-based procedure for generating a gene disruption construct. This method depends on DNA fragment fusion by the PCR technique and requires only two steps of PCR to obtain a sufficient amount of the gene disruption construct for one transformation experiment. The first step involves three separate PCR syntheses of a selectable marker cassette and the 5′- and 3′-regions of a target gene. Of the four primers used in amplification of the 5′- and 3′-regions of the target gene, two primers placed proximal to the site of the marker cassette are designed to have sequence tags complementary to the 5′- or 3′-side of the marker cassette. The two primers used in PCR synthesis of the marker cassette are complementary to the tagged primers. By fusion PCR, the 5′ and 3′ PCR products are linked to the marker cassette via the regions of tagged primers that overlap. A sufficient amount of the disruption construct can be directly amplified with the outermost primers. This method is simple, rapid and relatively inexpensive. In addition, there is the freedom of attaching long flanking regions to any selectable marker cassette.     

PCR fragmentation of DNA

A method has been developed to prepare random DNA fragments using PCR. First, two cycles are carried out at 16 degrees C with the Klenow's fragment and oligonucleotides (random primers) with random 3'-sequences and the 5'-constant part containing the site for cloning with the site-specific endonuclease. The random primers can link to any DNA site, and random DNA fragments are formed during DNA synthesis. During the second cycle, after denaturation of the DNA and addition of the Klenow's fragment, the random primers can link to newly synthesized DNA strands, and after DNA synthesis single-stranded DNA fragments are produced which have a constant primer sequence at the 5'-end and a complementary to it sequence at the 3'-end. During the third cycle, the constant primer is added and double-stranded fragments with the constant primer sequences at both ends are formed during DNA synthesis. Incubation for 1 h at 37 degrees C degrades the oligonucleotides used at the first stage due to endonuclease activity of the Klenow's fragment. Then routine PCR amplification is carried out using the constant primer. This method is more advantageous than hydrodynamic methods of DNA fragmentation widely used for "shotgun" cloning.     

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。     

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.     

基质金属蛋白酶组织抑制剂-2基因的人工合成与克隆

根据GenBank报道的基质金属蛋白酶组织抑制剂-2(TIMP-2)氨基酸序列和毕赤酵母偏爱密码子设计,通过化学方法合成得到适合在毕赤酵母中表达的目的TIMP-2基因序列,并将其克隆到质粒pPIC9中,构建了pPIC9-T2表达载体,PCR鉴定及测序结果表明得到了正确的TIMP-2基因序列。     

A PCR-based method for manipulation of the vaccinia virus genome that eliminates the need for cloning.

A general method is described for altering specific genes of vaccinia virus (VV). We demonstrate and evaluate the procedure by gene inactivation, using a dominant selectable marker in conjunction with recombinant polymerase chain reaction (PCR). Primers based on the sequence of the target gene enable amplification of flanking arms and their subsequent attachment to the gpt cassette that confers resistance to mycophenolic acid. Linear PCR constructs are transfected into cells infected with wild-type vaccinia virus. Mutant viruses with gpt inserted into the target gene by homologous recombination are then selected by growth in the presence of MPA. This technique was applied to the vaccinia virus thymidine kinase gene and compared to the traditional method of constructing gpt-containing plasmids by cloning. The PCR scheme was found to be highly efficient and could theoretically be used to insert any foreign DNA element into any nonessential target gene for which partial or complete sequence information is available. The procedure can potentially be used for a wide variety of genetic modifications, including the insertion of foreign genes, with poxviruses and other DNA viruses. Genomes of microorganisms, such as bacteria and yeast that can be transformed with linear DNA, are also candidates for manipulation by this methodology.     

Sequence specific generation of a DNA panhandle permits PCR amplification of unknown flanking DNA.

We present a novel method for the PCR amplification of unknown DNA that flanks a known segment directly from human genomic DNA. PCR requires that primer annealing sites be present on each end of the DNA segment that is to be amplified. In this method, known DNA is placed on the uncharacterized side of the sequence of interest via DNA polymerase mediated generation of a PCR template that is shaped like a pan with a handle. Generation of this template permits specific amplification of the unknown sequence. Taq (DNA) polymerase was used to form the original template and to generate the PCR product. 2.2 kb of the beta-globin gene, and 657 bp of the 5' flanking region of the cystic fibrosis transmembrane conductance regulator gene, were amplified directly from human genomic DNA using primers that initially flank only one side of the region amplified. This method will provide a powerful tool for acquiring DNA sequence information.     

Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence‐capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence‐capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence‐capture recovered 80%–90% of the control sample species. DNA sequence‐capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low‐cost, whereas DNA‐sequence capture for biodiversity assessment is still in its infancy, is more time‐consuming but provides more taxonomic assignments.     

A Method for Construction, Cloning and Expression of Intron-Less Gene from Unannotated Genomic DNA

The present century has witnessed an unprecedented rise in genome sequences owing to various genome-sequencing programs. However, the same has not been replicated with cDNA or expressed sequence tags (ESTs). Hence, prediction of protein coding sequence of genes from this enormous collection of genomic sequences presents a significant challenge. While robust high throughput methods of cloning and expression could be used to meet protein requirements, lack of intron information creates a bottleneck. Computational programs designed for recognizing intron–exon boundaries for a particular organism or group of organisms have their own limitations. Keeping this in view, we describe here a method for construction of intron-less gene from genomic DNA in the absence of cDNA/EST information and organism-specific gene prediction program. The method outlined is a sequential application of bioinformatics to predict correct intron–exon boundaries and splicing by overlap extension PCR for spliced gene synthesis. The gene construct so obtained can then be cloned for protein expression. The method is simple and can be used for any eukaryotic gene expression.     

Arbitrarily primed PCR fingerprinting of RNA.

Fingerprinting of RNA populations was achieved using an arbitrarily selected primer at low stringency for first and second strand cDNA synthesis. PCR amplification was then used to amplify the products. The method required only a few nanograms of total RNA and was unaffected by low levels of genomic double stranded DNA contamination. A reproducible pattern of ten to twenty clearly visible PCR products was obtained from any one tissue. Differences in PCR fingerprints were detected for RNAs from the same tissue isolated from different mouse strains and for RNAs from different tissues from the same mouse. The strain-specific differences revealed are probably due to sequence polymorphisms and should be useful for genetic mapping of genes. The tissue-specific differences revealed may be useful for studying differential gene expression. Examples of tissue-specific differences were cloned. Differential expression was confirmed for these products by Northern analysis and DNA sequencing uncovered two new tissue-specific messages. The method should be applicable to the detection of differences between RNA populations in a wide variety of situations.     

“Blind” mapping of genic DNA sequence polymorphisms in Lolium perenne L. by high resolution melting curve analysis

High resolution melting curve analysis (HRM) measures dissociation of double stranded DNA of a PCR product amplified in the presence of a saturating fluorescence dye. Recently, HRM proved successful to genotype DNA sequence polymorphisms such as SSRs and SNPs based on the shape of the melting curves. In this study, HRM was used for simultaneous screening and genotyping of genic DNA sequence polymorphisms identified in the Lolium perenne F2 mapping population VrnA. Melting profiles of PCR products amplified from previously published gene loci and from a novel gene putatively involved in vernalization response successfully discriminated genotypes in absence of allelic sequence information, and allowed to determine allele segregation in VrnA. Here we introduce the concept of “blind” mapping based on HRM as a powerful, fast and cheap method to map any DNA sequence polymorphisms without prior knowledge of allelic sequences in the key grassland species perennial ryegrass (Lolium perenne L.).     

A PCR-based approach to sequence the Candida tropicalis HSP90 gene

The heat shock protein 90 (hsp90) gene sequence is known to be highly conserved across the species barrier. A PCR-based method was thus utilised in an attempt to sequence the Candida tropicalis hsp90 gene. Primers for PCR were designed from conserved regions of the gene, which were identified by comparing the Saccharomyces cerevisiae and Candida albicans hsp90 gene sequences. Different sets of primers were designed to amplify and obtain overlapping DNA sequences of the C tropicalis gene. PCR was carried out on genomic DNA of Candidca tropicalis and the PCR products were cloned into suitable vector molecules for sequencing. In this way, a 2,070-basepair sequence of the C. tropicalis hsp90 gene was obtained. The PCR-based approach proved to be an easier method of obtain the sequence of a highly conserved gene, as compared to more conventional methods.     

重组PCR技术的应用及优化

重组PCR是通过DNA重叠序列的衔接作用,使多个DNA分子融合在一起的体外扩增技术。它使基因全序列的拼接、基因融合、基因破坏及启动子交换等DNA操作变得简单易行。如今重组PCR已成为DNA分析的有效利器。本研究通过重组PCR在分子进化、基因敲除及基因敲入、启动子研究和转基因植物转化载体的构建等方面的实际应用,分析了该技术的影响因素,并针对引物设计、DNA碱基重叠长度、温度参数等重要反应条件提出了优化方案。