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PCR-based gene synthesis with overlapping unisense-oligomers asymmetric extension supported by a simulator for oligonucleotide extension achieved 1 kbp dsDNA. 基于聚合酶链反应的重叠寡核苷酸-寡聚物不对称延伸基因合成在寡核苷酸延伸模拟器的支持下获得了1 kbp的dsDNA。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-06-01 DOI: 10.2144/btn-2022-0127
Yasunori Nishida, Kotetsu Kayama, Taichi Endoh, Kiwamu Hanazono, Gerry Amor Camer, Daiji Endoh
{"title":"PCR-based gene synthesis with overlapping unisense-oligomers asymmetric extension supported by a simulator for oligonucleotide extension achieved 1 kbp dsDNA.","authors":"Yasunori Nishida,&nbsp;Kotetsu Kayama,&nbsp;Taichi Endoh,&nbsp;Kiwamu Hanazono,&nbsp;Gerry Amor Camer,&nbsp;Daiji Endoh","doi":"10.2144/btn-2022-0127","DOIUrl":"https://doi.org/10.2144/btn-2022-0127","url":null,"abstract":"<p><p>We formulated a method to synthesize 1 kbp DNA fragments using 'oligomer unidirectional joining method' via asymmetric extension supported by a simulator for oligonucleotide extension (AESOE). In this study, trials were conducted on 41 sets of different genomic pieces of ten flaviviral genomes, and 31 bacterial 16s rRNA fragments with sizes ranging from 500 bases to 1.0 kbp. Synthetic gene production was found to be successful in all those sets. The synthesis method has three steps: the first step is a seven-linked AESOE, the second step is the linking of the 400-base fragments from the first step, and the third step is the final amplification. Our present approach is highly reproducible and may no longer require optimization of oligomer design.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9873865","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Gibson assembly interposition improves amplification efficiency of long DNA and multifragment overlap extension PCR. 吉布森装配插入法提高了长DNA和多片段重叠延伸PCR的扩增效率。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-06-01 DOI: 10.2144/btn-2023-0012
Junyi Liu, Fangyin Liu, Xueer Luo, Ming Chen, Chengjun Wang, Liuyue Wang, Huabo Chen
{"title":"Gibson assembly interposition improves amplification efficiency of long DNA and multifragment overlap extension PCR.","authors":"Junyi Liu,&nbsp;Fangyin Liu,&nbsp;Xueer Luo,&nbsp;Ming Chen,&nbsp;Chengjun Wang,&nbsp;Liuyue Wang,&nbsp;Huabo Chen","doi":"10.2144/btn-2023-0012","DOIUrl":"https://doi.org/10.2144/btn-2023-0012","url":null,"abstract":"<p><p>For difficult overlap extension PCR, a Gibson assembly process was inserted between the two PCR rounds to facilitate the formation of complete gene templates at a moderate temperature. That is, after amplifying each DNA fragment, they were preluded by a Gibson assembly process in equal proportion. Then, the assembled mixture was used as a template for the second PCR round. This idea was tested and verified by taking the cloning example of a single and a double site mutation of the retinoblastoma gene. This scheme associates overlap extension PCR with Gibson assembly exquisitely, significantly improving gene amplification efficiency, particularly in the fusion of long genes and multifragments using overlap extension PCR.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10231372","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Direct-current electric field stimulation promotes proliferation and maintains stemness of mesenchymal stem cells. 直流电刺激可促进间充质干细胞增殖，维持干细胞的干性。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-06-01 DOI: 10.2144/btn-2022-0112
Mengchang Liu, Defu Xie, Huizhen Zeng, Ning Zhai, Lan Liu, Hong Yan
{"title":"Direct-current electric field stimulation promotes proliferation and maintains stemness of mesenchymal stem cells.","authors":"Mengchang Liu,&nbsp;Defu Xie,&nbsp;Huizhen Zeng,&nbsp;Ning Zhai,&nbsp;Lan Liu,&nbsp;Hong Yan","doi":"10.2144/btn-2022-0112","DOIUrl":"https://doi.org/10.2144/btn-2022-0112","url":null,"abstract":"<p><p>Mesenchymal stem cells are frequently utilized in the study of regenerative medicine. Electric fields (EFs) influence many biological processes, such as cell proliferation, migration and differentiation. In the present study, a novel device capable of delivering a direct current of EF stimulation to cells cultured <i>in vitro</i> is described. This bioreactor was customized to simultaneously apply a direct-current EF to six individual cell culture wells, which reduces the amount of experimental time and minimizes cost. In testing the device, adipose-derived mesenchymal stem cells stimulated with an EF in the bioreactor exhibited a greater cell proliferation rate while retaining stemness. The results provide a unique perspective on adipose-derived mesenchymal stem cell proliferation, which is needed for tissue engineering and regenerative medicine.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9873386","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
The path to solving the protein folding problem. 解决蛋白质折叠问题的途径。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-05-01 DOI: 10.2144/btn-2023-0031
Jenny Straiton
{"title":"The path to solving the protein folding problem.","authors":"Jenny Straiton","doi":"10.2144/btn-2023-0031","DOIUrl":"https://doi.org/10.2144/btn-2023-0031","url":null,"abstract":"<p><p>[Formula: see text] With advances in imaging technologies and the development of artificial intelligence-based predictive software, has the protein folding problem finally been solved?</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10004374","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Target-seq: single workflow for detection of genome integration site, DNA translocation and off-target events. Target-seq:检测基因组整合位点、DNA易位和脱靶事件的单一工作流程。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-05-01 DOI: 10.2144/btn-2023-0013
Pei-Zhong Tang, Bo Ding, Christopher Reyes, David Papp, Jason Potter
{"title":"Target-seq: single workflow for detection of genome integration site, DNA translocation and off-target events.","authors":"Pei-Zhong Tang,&nbsp;Bo Ding,&nbsp;Christopher Reyes,&nbsp;David Papp,&nbsp;Jason Potter","doi":"10.2144/btn-2023-0013","DOIUrl":"https://doi.org/10.2144/btn-2023-0013","url":null,"abstract":"<p><p>Designed donor DNA delivery through viral or nonviral systems to target loci in the host genome is a critical step for gene therapy. Adeno-associated virus and lentivirus are leading vehicles for <i>in vivo</i> and <i>ex vivo</i> delivery of therapeutic genes due to their high delivery and editing efficiency. Nonviral editing tools, such as CRISPR/Cas9, are getting more attention for gene modification. However, there are safety concerns; for example, tumorigenesis due to off-target effects and DNA rearrangement. Analysis tools to detect and characterize on-target and off-target genome modification post editing in the host genome are pivotal for evaluating the success and safety of gene therapy. We developed Target-seq combined with different analysis tools to detect the genome integration site, DNA translocation and off-target events.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10002812","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
High-quality total RNA extraction from early-stage lamprey embryos. 从早期七鳃鳗胚胎中提取高质量的总RNA。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-05-01 DOI: 10.2144/btn-2023-0004
Fumiaki Sugahara, Juan Pascual-Anaya
{"title":"High-quality total RNA extraction from early-stage lamprey embryos.","authors":"Fumiaki Sugahara,&nbsp;Juan Pascual-Anaya","doi":"10.2144/btn-2023-0004","DOIUrl":"https://doi.org/10.2144/btn-2023-0004","url":null,"abstract":"<p><p>High-purity total RNA extraction from animal embryos is essential for transcriptome analyses. lampreys, together with hagfish, are the only extant jawless vertebrates or cyclostomes and are thus key organisms for EvoDevo studies. However, extracting uncontaminated RNA from early-stage embryos remains challenging. RNA does not bind to the silica membrane in filter-based extractions, significantly reducing yields; and ethanol/isopropanol precipitation methods lead to contaminants, bringing down the optical density (OD) 260/280 ratio. The RNA extraction protocol was modified using precentrifugation and adding salts before isopropanol precipitation. This modification significantly increased RNA yield, removed contaminants and improved RNA integrity. Egg membrane sources were suspected to cause RNA purification problems because low-quality extraction does not occur in posthatching embryos.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10003328","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Instant Pot for antigen retrieval: a simple, safe and economical method for use in immunohistochemistry. 用于抗原回收的速溶锅：一种用于免疫组化的简单、安全和经济的方法。
IF 2.2 4区 工程技术
BioTechniques Pub Date : 2023-05-01 Epub Date: 2023-05-18 DOI: 10.2144/btn-2022-0043
Nolan Kearns, Karli Norville, John G Frelinger
{"title":"Instant Pot for antigen retrieval: a simple, safe and economical method for use in immunohistochemistry.","authors":"Nolan Kearns, Karli Norville, John G Frelinger","doi":"10.2144/btn-2022-0043","DOIUrl":"10.2144/btn-2022-0043","url":null,"abstract":"<p><p>Here, the authors report a simple method to perform antigen retrieval using a commonly available commercial Instant Pot<sup>®</sup> for immunohistochemistry. It provides a validated alternative to previous antigen retrieval methods that employ water baths, microwave ovens or scientific-grade pressure cookers. The Instant Pot can be set to obtain a variety of desired temperatures and is straightforward to use, making it extremely amenable to optimization. The Instant Pot method is an easy, safe and inexpensive alternative means to perform immunohistochemistry on formalin-fixed paraffin-embedded sections. It has been validated using several different monoclonal antibodies including ones directed against cell surface or intracellular antigens. As a result, it should be useful for a variety of research labs as well as undergraduate laboratory courses.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/21/7d/btn-74-237.PMC10265304.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9993450","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
An optimized TRIzol-based method for isolating RNA from adipose tissue. 基于trizol的脂肪组织RNA分离方法的优化。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-05-01 DOI: 10.2144/btn-2022-0120
Hongwei Zhang, Yaoming Liu, Bingcheng Yu, Rong Lu
{"title":"An optimized TRIzol-based method for isolating RNA from adipose tissue.","authors":"Hongwei Zhang,&nbsp;Yaoming Liu,&nbsp;Bingcheng Yu,&nbsp;Rong Lu","doi":"10.2144/btn-2022-0120","DOIUrl":"https://doi.org/10.2144/btn-2022-0120","url":null,"abstract":"<p><p>High-quality RNA isolation from recalcitrant adipose tissue with high lipid content and low cell numbers is difficult. Many studies have made efforts to optimize methods for isolating RNA from adipose tissue through combinations of column-based kits and phenol-chloroform methods, or through in-house protocols. However, the considerable complexity of these protocols and the various kits/materials required hamper their wide use. Herein, we describe an optimized protocol based on TRIzol reagent, which is the most accessible ready-to-use reagent for nucleic acid and/or protein isolation in laboratories. This article provides a step-by-step protocol yielding sufficient and qualified RNA from lipid-rich specimens for downstream applications.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9684601","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Site-specific analysis of ribosomal 2'O-methylation by quantitative reverse transcription PCR under low deoxynucleotide triphosphate concentrations. 低三磷酸脱氧核苷酸浓度下核糖体2' o甲基化的定量反转录PCR位点特异性分析。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-05-01 DOI: 10.2144/btn-2022-0122
Daniela Barros-Silva, Johan Tsui, Carmen Jerónimo, Guido Jenster, Elena S Martens-Uzunova
{"title":"Site-specific analysis of ribosomal 2'O-methylation by quantitative reverse transcription PCR under low deoxynucleotide triphosphate concentrations.","authors":"Daniela Barros-Silva,&nbsp;Johan Tsui,&nbsp;Carmen Jerónimo,&nbsp;Guido Jenster,&nbsp;Elena S Martens-Uzunova","doi":"10.2144/btn-2022-0122","DOIUrl":"https://doi.org/10.2144/btn-2022-0122","url":null,"abstract":"<p><p>Ribose 2'O-methylation (Nm, ribomethylation) is the most abundant RNA modification present in rRNA. It has been shown that alterations in ribosomal 2'O-methylation at individual Nm sites likely reflect regulated cellular processes. Although several analytical approaches for Nm detection and profiling have been developed, a simple and affordable method for the screening and measurement of individual Nm sites in large numbers of tissue samples is required to examine their potential for clinical translation. Here, we describe a new quantitative reverse transcription PCR-based method that can sensitively assess ribomethylation levels at specific rRNA sites at single-nucleotide resolution in low input amounts of total RNA.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9987171","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Comparison of magnetic bead and rapid swab RNA extraction methods for detecting rabbit hemorrhagic disease virus 2 in rabbit liver samples. 磁珠和快速拭子RNA提取法检测兔肝脏出血性疾病病毒2的比较。
IF 2.7 4区 工程技术
BioTechniques Pub Date : 2023-04-01 DOI: 10.2144/btn-2022-0022
Erik Hofmeister, Kathryn Griffin, Hon Ip
{"title":"Comparison of magnetic bead and rapid swab RNA extraction methods for detecting rabbit hemorrhagic disease virus 2 in rabbit liver samples.","authors":"Erik Hofmeister,&nbsp;Kathryn Griffin,&nbsp;Hon Ip","doi":"10.2144/btn-2022-0022","DOIUrl":"https://doi.org/10.2144/btn-2022-0022","url":null,"abstract":"<p><p>We compared a bead RNA extraction method with a one-tube method that required only a heat block and ice. RNA was first extracted from liver samples from nine rabbits dying from rabbit hemorrhagic disease virus 2 (RHDV2) using magnetic beads, and RT-PCR was used to detect RHDV2 sequence. Following freezing, RNA was extracted a second time using the SwiftX™ Swabs Viral RNA Extraction Reagent. RHDV2 was detected in all nine samples. Cycle threshold values were higher in the RT-PCR following SwiftX extraction (mean: 3.79), indicating that the second extraction method resulted in approximately a 1 log<sub>10</sub> reduction in sensitivity. A second freeze-thaw for the samples and less tissue extracted using SwiftX may have contributed additionally to the loss in sensitivity.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9629606","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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