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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. 相似文献
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Pingping Zhang Yingying Ding Wenting Liao Qiuli Chen Huaqun Zhang Peipei Qi Ting He Jinhong Wang Songhua Deng Tianyue Pan Hao Ren Wei Pan 《Gene》2013
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. 相似文献
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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. 相似文献
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Pu Yan Xinzheng Gao Peng Zhou 《Biochemical and biophysical research communications》2009,383(4):464-468
Hairpin RNA (hpRNA) is commonly used for gene-function exploration and gene engineering. In this study, a novel method was developed to construct intron-containing hairpin RNA (ihpRNA) rapidly and efficiently based on Overlap Extension PCR (OE-PCR). This method, Mixed One-step OE-PCR (MOOE-PCR), can amplify two inverted repeats of DNA fragments and a spliceable intron in parallel, and then assemble them to generate ihpRNA constructs in the same tube without the purification of intermediate products. This method required a PCR process of 38-40 cycles and ordinary PCR reagents. A total of 10 ihpRNA constructs were amplified successfully using this method, with the stems ranging from 50 bp to 484 bp in length. Our results suggest that this novel method is a useful strategy for constructing ihpRNA. 相似文献
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