A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences

Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression. Here, we report a simple, high-fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are ~500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Compared with the previously published methods, the PTDS method is rapid (5–7 days) and suitable for synthesizing long segments of DNA (5–6 kb) with high G + C contents, repetitive sequences or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures. Using the newly developed PTDS method, we have successfully obtained several genes of interest with sizes ranging from 1.0 to 5.4 kb.     

Total synthesis of multi-kilobase DNA sequences from oligonucleotides

A method for synthesizing DNA from 40-mer oligonucleotides, which we used to generate a 32-kb DNA fragment, is explained. DNA sequences are synthesized as approximately 500 bp fragments (synthons) in a two-step PCR reaction and cloned using ligation-independent cloning (LIC). Synthons are then assembled into longer full-length sequences in a stepwise manner. By initially synthesizing smaller fragments (synthons), the number of clones sequenced is low compared with synthesizing complete multi-kilobase DNA sequences in a single step. LIC eliminates the need for purification of fragments before cloning, making the process amenable to high-throughput operation and automation. Type IIs restriction enzymes allow seamless assembly of synthons without placing restrictions on the sequence being synthesized. Synthetic fragments are assembled in pairs to generate the final construct using vectors that allow selection of desired clones with two unique antibiotic resistance markers, and this eliminates the need for purification of fragments after digestion with restriction endonucleases.     

Enrichment of meiotic recombination hotspot sequences by avidin capture technology

About 40% of the hotspots for meiotic recombination contain the degenerate consensus sequence 5′-CCNCCNTNNCCNC-3′. Here we present a novel protocol for enriching hotspot sequences from digested genomic DNA by using biotinylated oligonucleotides and streptavidin-coated magnetic beads. The captured hotspots can be released by simple digestion with restriction enzymes for subsequent characterization by second generation sequencing or PCR. The capture protocol specifically enriches hotspot sequences, judged by using fluorophore-conjugated synthetic oligonucleotides and synthetic double-stranded oligonucleotides in combination with PCR. The capture protocol enriches single-stranded DNA, denatured double-stranded DNA, and large fragments (> 3000 bp) of digested plasmid DNA with good efficacy. No false positive and false negatives were detected when enriching digested DNA from human cell cultures and primary human cells. The protocol can probably be adapted to enriching sequences other than the hotspot sequence by altering the sequence in the capture oligonucleotide. We intend to apply this protocol in studies assessing effects of micronutrient status on meiotic recombination events in human sperm.     

基于PCR的长片段DNA精确合成及应用

长片段DNA的组装及基于PCR的长片段DNA的精确合成(PAS)是基因异源表达时简单快速合成长片段DNA的方法,适于合成含有较高GC含量、重复序列和复杂二级结构的长片段DNA分子(5～6 kb)。简要综述了PAS技术用于长片段DNA序列精确合成的基本原理、操作步骤、常见问题和解决方法,以及该技术的应用。     

Patch oligodeoxynucleotide synthesis (POS): a novel method for synthesis of long DNA sequences and full-length genes

Synthesis of long DNA fragments is often associated with mutations and requires multiple DNA manipulation steps. A novel DNA synthesis method, referred to as patch oligodeoxynucleotide synthesis (POS) to assembly long DNA fragments is presented here. This method involves connection of two types of oligodeoxynucleotides: long constructional oligonucleotides (COs) and short patch oligonucleotides (POs). Long COs were connected by a ligase with the aid of POs, which were complementary to both adjacent COs to help remove secondary structures during assembly. The partial double-stranded DNA template that was formed was then amplified by PCR. Accordingly, we synthesized SV40 polyadenylation signal sequences (187 bp), a codon-optimized yellow fluorescent protein gene (678 bp), and Rattus norvegicus catenin β1 (2,352 bp). This presented method can be broadly applied to synthesize DNA fragments of varying lengths with great convenience.     

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.     

Cloning genomic sequences using long-range PCR

Protocols are presented for preparing DNA from a genomic library in λ phage and for synthesizing genomic fragments using PCR with nested vector- and gene-specific primers and linker-primers. Library DNA, isolated fromE. coli liquid lysates by a simple protocol, is used as template in PCR following a commercial protocol. The method produces library DNA sufficient for several hundred PCRs, incorporates nested primers to reduce nonspecific product formation, and enables the synthesis of linker-containing DNA fragments containing selected restriction sites to simplify subsequent cloning. The isolation of 5′ upstream sequences of three different arabidopsis genes by this methodod is described.     

Correcting errors in synthetic DNA through consensus shuffling

Although efficient methods exist to assemble synthetic oligonucleotides into genes and genomes, these suffer from the presence of 1–3 random errors/kb of DNA. Here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding protein (MutS). PCR assembly of the remaining fragments yields a new population of full-length sequences enriched for the consensus sequence of the input population. We show that two iterations of consensus shuffling improved a population of synthetic green fluorescent protein (GFPuv) clones from ~60 to >90% fluorescent, and decreased errors 3.5- to 4.3-fold to final values of ~1 error per 3500 bp. In addition, two iterations of consensus shuffling corrected a population of GFPuv clones where all members were non-functional, to a population where 82% of clones were fluorescent. Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences.     

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.     

A rapid PCR-based method for identification of four important Candida species

The rapid detection and identification of Candida species in clinical laboratories are extremely important for the management of patients with hematogenous candidosis. Currently available culture and biochemical methods for detection and identification of Candida species are time-consuming. This study describes the use of a simple and rapid PCR method using species-specific oligonucleotides for the detection of clinical isolates of Candida species. These species-specific oligonucleotides are complementary to unique sequences within the intergenic transcribed spacer 2, located in between the 5.8S and 28S ribosomal DNA, and generated DNA fragments by both the conventional and hemi-nested PCR reactions. Conventional PCR produced a single DNA fragment of variable size in all isolates, while the hemi-nested PCR produced two discrete DNA fragments, both with the expected sizes of 111bp/57bp (C. albicans), 84bp/42bp (C. glabrata), 94bp/45bp (C. krusei) and 95bp/49bp (C. parapsilosis). In conclusion, the PCR-based method described in this study is fast and specific for the identification of clinically important Candida species.     

Oligonucleotide-primed in situ DNA synthesis (PRINS): a method for chromosome mapping, banding, and investigation of sequence organization

Oligonucleotides were annealed to complementary sequences in fixed human metaphase chromosomes and extended with DNA polymerase. The newly synthesized fragments were labeled by incorporating bio-11-dUTP instead of TTP, and the sites of synthesis were detected by immunocytochemistry, using fluorochromes as the reporter molecules. We have obtained clear localization with oligonucleotides from alphoid (centromeric sequences), simple sequence (satellite) DNAs, a variety of Alu-dispersed repeated sequences, and oligonucleotides derived from the Tetrahymena and Trypanosoma telomere-specific sequences. The simple sequence and alphoid oligonucleotides gave results at least comparable to those obtained using the whole molecule as a probe for in situ hybridization, whereas the Alu oligonucleotides produced a diversity of results which depended on the absolute length and location of the oligonucleotide within the Alu sequence. The telomere-specific oligomers also produced a variety of results. The G-rich Trypanosoma oligomer and its complementary C-rich sequence produced strong telomeric signals and some interstitial signals on mouse chromosomes, but only weak telomeric signals on human chromosomes. The G-rich Tetrahymena oligomer produced detectable telomeric signals on human chromosomes. The technique appears to be a valuable extension of present tools for mapping and examining the organization of DNA sequences within chromosomes.     

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.     

PCR- and ligation-mediated synthesis of marker cassettes with long flanking homology regions for gene disruption in Saccharomyces cerevisiae.

We developed a novel method for synthesizing marker-disrupted alleles of yeast genes. The first step is PCR amplification of two sequences located upstream and downstream of the reading frame to be disrupted. Due to the addition of non-specific single A overhangs by Taq DNA polymerase, each PCR product can be ligated with a marker DNA which has T residues at its 3' ends. After amplification of individual ligation products through the second PCR, both products are mixed and annealed, and the single strand is converted to a double strand by an extension reaction. The final step is PCR amplification of the fragment composed of a selectable marker and two flanking sequences with the outermost primers. This method is rapid and needs only short oligonucleotides as primers.     

Improved PCR-based gene synthesis method and its application to the Citrobacter freundii phytase gene codon modification

Gene synthesis technologies provide a powerful tool for increasing protein expression through codon optimization and gene modification. Here we describe an improved PCR-based gene synthesis technology, which is accurate, simple and cheap. The improved PCR-based gene synthesis (IPS) method consists of two steps. The first one is the synthesis of 300-400 bp fragments by PCR reaction with Pfu DNA polymerase from 60-mer and 30-mer oligonucleotides with a 15 bp overlap. The second one is assembling of fragments from the first step into the full-length gene by PCR reaction. Using this approach, we have successfully synthesized a modified phytase gene with 1256 bp in length with optimal codons for expression in Pichia pastoris. P. pastoris strain that expressed the modified phytase gene (phyA-mod) showed a 50% increase in phytase activity level. In addition, we propose an inexpensive method for error correction, based on overlap-extension PCR (OE-PCR).     

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.     

利用DNA芯片技术高效合成OstreococcusTauri叶绿体基因组

目的：21世纪以来,随着合成生物学的高速发展及其所遇到的问题,开发下一代DNA合成技术已经成为了必然趋势。基因芯片技术和DNA大片段组装技术是建立下一代DNA合成平台的关键技术力量。方法：为了开发具有工业化标准的DNA芯片一基因组合成平台,我们首次利用电化学DNA芯片和DNA大片段组装技术合成了72kb的Ostreococcusmud的全叶绿体基因组。结果：首先,我们使用电化学DNA芯片合成仪合成了564条150bp的OligoMix,并成功扩增分离了其中96％的Oligo序列,剩下的基因组序列是通过传统的固相亚磷酰胺三脂合成法合成。在此基础上,我们利用DNA重组技术将564条150bpOligo片段分三步克隆到了一个pGSYN系统。通过高通量测序,我们证实叶绿体基因组被成功地人工合成。整个合成成本大约是目前传统基因合成成本的10％．20％。结论：研究证实基因芯片技术和DNA大片段组装技术的应用是能够明显的降低现阶段基因组合成工艺的成本。新技术的成熟推广和成本的有效控制也会进一步加速科学家对基因组功能的深入研究以及合成生物学的质的飞跃。     

Genome mapping by arbitrary amplification of yeast artificial chromosomes

Several methods have been described for using the polymerase chain reaction (PCR) to isolate fragments of DNA for genome mapping. We have developed an approach for isolating discrete fragments by amplifying DNA with single oligonucleotides (10-mers) with arbitrarity selected sequences. The method is rapid and technically simple. We isolated fragments from a contig of three yeast artificial chromosomes (YACs) from the human Xq28 chromosomal region. We purified YACs yWXD 37, yWXD348, and yWXD705 from a preparative pulsed field gel. Amplifications of each YAC were performed with single 10-mers as the PCR primers and the products were visualized on agarose gels. These fragments have been successfully used as hybridization probes against Southern blots containing the YACs and against blots containing human genomic DNA and somatic cell hybrids containing Xq28 as their only human constituent. The results have been concordant with the known order of the YACs. We have also successfully combined 10-mers with primers derived from vector arm sequences to isolate YAC ends. We discuss several uses of this method in comparative mapping and in filling in gaps in physical and genetic maps.     

Cloning of the Escherichia coli gene for the stringent starvation protein

Summary In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with ³²P at their 5-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were coloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.Abbreviations SSP stringent starvation protein - PTH phenylthiohydantoin     

一种简单快速克隆IL-6信号转导相关基因的方法

细胞因子作用于受体时的一个重要结果是诱导基因表达。为了克隆与IL-6诱导相关的基因,我们利用一个快速的改良DD-PCR方法,分离并检测了IL-6诱导和未诱导的U937细胞的差异表达基因。用三个完全变性的6—mer引物进行反转录,用2或3个较长的随机引物进行PCR扩增,扩增产物很在2%琼脂糖凝胶电泳上分离,之后回收差异片段并直接用于克隆和测序。在研究中,获得了7个不同的EST,序列分析表明其中2个EST可能是与细胞信号转导相关的新基因片段;反向Northern杂交证实它们是与IL-6作用相关的差异表达基因。     

Single restriction enzyme-assisted megaprimer polymerase chain reaction to fuse two DNA sequences on separate cloning vectors

We describe a simple and versatile method to fuse two DNA sequences on separate cloning vectors in a single polymerase chain reaction (PCR). The method, termed restriction enzyme-assisted megaprimer PCR (REM–PCR), requires that the two cloning vectors share a common sequence and that the DNA sequences to be fused are cloned in the same orientation with respect to the common sequence. Fusion of the two sequences is achieved by mutual priming at the common sequence between two DNA fragments that were generated by restriction enzyme and linearly amplified by repetitive priming in the PCR reaction mixture.