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一种方便、无PCR过程的DNA shuffling方法
科学通报 , 2004,
Abstract: 目前大多数的DNA shuffling方法都需要PCR过程.由于一些限制内切酶的识别位点与切割位点不重叠而产生不同的黏性末端,当再连接的时候,同源片段之间就可以混合起来按照正确顺序组装成新的嵌合体分子,利用这种性质,构建了一种新的、无PCR过程的、简便的shuffling方法.
Directed Evolution of D-lactonohydrolase by Error Prone PCR and DNA Shuffling

LIU Zhi-Qiang,SUN Zhi-Hao,ZHENG Pu,LENG Yong,QIAN Jia-Nan,

生物工程学报 , 2005,
Abstract: D-lactonohydrolase is useful in the procedure of resolution of racemic pantolactone to produce D-pantolactone, but the activity and stability under low pH of the wild type enzyme is not satisfactory enough to be applied to industrial production. The expected properties of wild type enzyme were enhanced by directed evolution. According to the formation of products and pH indicators, a screening system was designed. After three sequential error prone PCR and one round DNA shuffling followed by screening, Mut E-861, the best mutant with improved activity and stability under low pH situation was obtained. Gene analysis of the Mut E-861 mutant indicated that the mutant enzyme had A352C, G721A mutations and a silent mutation of position 1038. Moreover, the activity and stability of Mut E-861 were determined. The results showed that the activity of this mutant was 5.5-fold higher than that of wild type, and the stability under low pH was improved at no expense of D-lactonohydrolase activity. After incubated at pH 6.0 and pH 5.0 the activity of D-lactonohydrolase could be retained 75% to 50%, however, compared with 40% to 20% for wild type.
A novel three primers PCR (TP-PCR) method to obtain recombinant DNA molecule independent of restriction enzyme
Chaoyang Deng,Guisheng Song,Junwang Xu,Zhen Zhu
Chinese Science Bulletin , 2002, DOI: 10.1360/02tb9448
Abstract: In this note, we report a novel and efficient three primers PCR (TP-PCR) method to rapidly generate recombinant DNA molecule at precise junction between two arbitrary DNA fragments. TP-PCR method is characterized by its reaction system with two templates and three primers, which can produce a recombinant DNA molecule in one PCR reaction. The main advantages of this method are the independence of sequences at the recombination site, the rapidness, and the easy establishment of adequate conditions. This method has been successfully applied to constructing a fusion protein gene, sck gene.
Development and Functional Analysis of Novel Genetic Promoters Using DNA Shuffling, Hybridization and a Combination Thereof  [PDF]
Rajiv Ranjan, Sunita Patro, Bhubaneswar Pradhan, Alok Kumar, Indu B. Maiti, Nrisingha Dey
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0031931
Abstract: Background Development of novel synthetic promoters with enhanced regulatory activity is of great value for a diverse range of plant biotechnology applications. Methodology Using the Figwort mosaic virus full-length transcript promoter (F) and the sub-genomic transcript promoter (FS) sequences, we generated two single shuffled promoter libraries (LssF and LssFS), two multiple shuffled promoter libraries (LmsFS-F and LmsF-FS), two hybrid promoters (FuasFScp and FSuasFcp) and two hybrid-shuffled promoter libraries (LhsFuasFScp and LhsFSuasFcp). Transient expression activities of approximately 50 shuffled promoter clones from each of these libraries were assayed in tobacco (Nicotiana tabacum cv. Xanthi) protoplasts. It was observed that most of the shuffled promoters showed reduced activity compared to the two parent promoters (F and FS) and the CaMV35S promoter. In silico studies (computer simulated analyses) revealed that the reduced promoter activities of the shuffled promoters could be due to their higher helical stability. On the contrary, the hybrid promoters FuasFScp and FSuasFcp showed enhanced activities compared to F, FS and CaMV 35S in both transient and transgenic Nicotiana tabacum and Arabidopsis plants. Northern-blot and qRT-PCR data revealed a positive correlation between transcription and enzymatic activity in transgenic tobacco plants expressing hybrid promoters. Histochemical/X-gluc staining of whole transgenic seedlings/tissue-sections and fluorescence images of ImaGene Green? treated roots and stems expressing the GUS reporter gene under the control of the FuasFScp and FSuasFcp promoters also support the above findings. Furthermore, protein extracts made from protoplasts expressing the human defensin (HNP-1) gene driven by hybrid promoters showed enhanced antibacterial activity compared to the CaMV35S promoter. Significance/Conclusion Both shuffled and hybrid promoters developed in the present study can be used as molecular tools to study the regulation of ectopic gene expression in plants.
Directed DNA Shuffling of Retrovirus and Retrotransposon Integrase Protein Domains  [PDF]
Xiaojie Qi, Edwin Vargas, Liza Larsen, Whitney Knapp, G. Wesley Hatfield, Richard Lathrop, Suzanne Sandmeyer
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0063957
Abstract: Chimeric proteins are used to study protein domain functions and to recombine protein domains for novel or optimal functions. We used a library of chimeric integrase proteins to study DNA integration specificity. The library was constructed using a directed shuffling method that we adapted from fusion PCR. This method easily and accurately shuffles multiple DNA gene sequences simultaneously at specific base-pair positions, such as protein domain boundaries. It produced all 27 properly-ordered combinations of the amino-terminal, catalytic core, and carboxyl-terminal domains of the integrase gene from human immunodeficiency virus, prototype foamy virus, and Saccharomyces cerevisiae retrotransposon Ty3. Retrotransposons can display dramatic position-specific integration specificity compared to retroviruses. The yeast retrotransposon Ty3 integrase interacts with RNA polymerase III transcription factors to target integration at the transcription initiation site. In vitro assays of the native and chimeric proteins showed that human immunodeficiency virus integrase was active with heterologous substrates, whereas prototype foamy virus and Ty3 integrases were not. This observation was consistent with a lower substrate specificity for human immunodeficiency virus integrase than for other retrovirus integrases. All eight chimeras containing the Ty3 integrase carboxyl-terminal domain, a candidate targeting domain, failed to target strand transfer in the presence of the targeting protein, suggesting that multiple domains of the Ty3 integrase cooperate in this function.
An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents  [PDF]
Shy-Shin Chang, Hsung-Ling Hsu, Ju-Chien Cheng, Ching-Ping Tseng
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0020303
Abstract: Background Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found. Methodology/Principal Findings We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3′-end complementary to the template bacterial sequence and a 5′-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10–100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species. Conclusions/Significance Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology laboratories.
Anticipatory evolution and DNA shuffling
Jamie M Bacher, Brian D Reiss, Andrew D Ellington
Genome Biology , 2002, DOI: 10.1186/gb-2002-3-8-reviews1021
Abstract: Proteins are machines created by evolution, but it is unclear just how finely evolution has guided their sequence, structure, and function. It is undoubtedly true that individual mutations in a protein affect both its structure and its function and that such mutations can be fixed during evolutionary history, but it is also true that there are other elements of protein sequence that have been acted upon by evolution. For example, the genetic code appears to be laid out so that mutations and errors in translation are minimally damaging to protein structure and function [1]. Could the probability that a beneficial mutation is found and fixed in the population also have been manipulated during the course of evolution, so that the proteins we see today are more capable of change than the proteins that may have been cobbled together following the 'invention' of translation? Have proteins, in fact, evolved to evolve? There is already some evidence that bacteria are equipped to evolve phenotypes that are more capable of further adaptation (reviewed in [2,3,4]). For example, mutator [5] and hyper-recombinogenic [6] strains arise as a result of selection experiments. The development of DNA shuffling (reviewed in [7,8]) and the appearance of several recent papers using this technique [9,10,11] provide us with a surprising new opportunity to ask and answer these fundamental questions at the level of individual genes, and perhaps even genomes.DNA shuffling, a method for in vitro recombination, was developed as a technique to generate mutant genes that would encode proteins with improved or unique functionality [12,13]. It consists of a three-step process that begins with the enzymatic digestion of genes, yielding smaller fragments of DNA. The small fragments are then allowed to randomly hybridize and are filled in to create longer fragments. Ultimately, any full-length, recombined genes that are recreated are amplified via the polymerase chain reaction. If a series of alleles o
Exponential Megapriming PCR (EMP) Cloning—Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints  [PDF]
Alexander Ulrich, Kasper R. Andersen, Thomas U. Schwartz
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0053360
Abstract: We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.
A Novel Low Temperature PCR Assured High-Fidelity DNA Amplification  [PDF]
Guofan Hong,Sin Hang Lee,Shichao Ge,Shaoxia Zhou
International Journal of Molecular Sciences , 2013, DOI: 10.3390/ijms140612853
Abstract: As previously reported, a novel low temperature (LoTemp) polymerase chain reaction (PCR) catalyzed by a moderately heat-resistant (MHR) DNA polymerase with a chemical-assisted denaturation temperature set at 85 °C instead of the conventional 94–96 °C can achieve high-fidelity DNA amplification of a target DNA, even after up to 120 PCR thermal cycles. Furthermore, such accurate amplification is not achievable with conventional PCR. Now, using a well-recognized L1 gene segment of the human papillomavirus (HPV) type 52 (HPV-52) as the template for experiments, we demonstrate that the LoTemp high-fidelity DNA amplification is attributed to an unusually high processivity and stability of the MHR DNA polymerase whose high fidelity in template-directed DNA synthesis is independent of non-existent 3'–5' exonuclease activity. Further studies and understanding of the characteristics of the LoTemp PCR technology may facilitate implementation of DNA sequencing-based diagnostics at the point of care in community hospital laboratories.
High-throughput real-time PCR-based genotyping without DNA purification
Anastasia Fedick, Jing Su, Chaim Jalas, Nathan R Treff
BMC Research Notes , 2012, DOI: 10.1186/1756-0500-5-573
Abstract: The performances of either DNA purified from blood or the same blood samples without DNA purification were evaluated through qPCR-based genotyping. First, 60 different mutations prevalent in the Ashkenazi Jewish population were genotyped in 12 Ashkenazi Jewish individuals using the QuantStudio?12K Flex Real-Time PCR System. Genotyping directly from blood gave a call rate of 99.21%, and an accuracy of 100%, while the purified DNA gave a call rate of 92.49%, and an accuracy of 99.74%. Although no statistical difference was found for these parameters, an F test comparing the standard deviations of the wild type clusters for the two different methods indicated significantly less variation when genotyping directly from blood instead of after DNA purification. To further establish the ability to perform high-throughput qPCR based genotyping directly from blood, 96 individuals of Ashkenazi Jewish decent were genotyped for the same 60 mutations (5,760 genotypes in 5 hours) and resulted in a call rate of 98.38% and a diagnostic accuracy of 99.77%.This study shows that accurate qPCR-based high-throughput genotyping can be performed without DNA purification. The direct use of blood may further expedite the entire genotyping process, reduce costs, and avoid tracking errors which can occur during sample DNA purification.Many advances have been made in the field of genetics, including progress in high-throughput genotyping. The potential turnaround time due to the improved speed of genotyping however, is hindered by the slow upstream step of extracting genomic (g)DNA from blood samples. Manual or automated extraction of gDNA from blood can take at least several hours, without including the time it then takes to normalize each individual sample. Additionally, different volumes of blood are needed for different extraction methods, which are not always obtainable. Commercially available products from multiple vendors now allow for samples to be quickly prepared for genotyping withou
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