Current Protocols in Molecular Biology最新文献_第8页

Overview of Next-Generation Sequencing Technologies 新一代测序技术概述

Current Protocols in Molecular Biology Pub Date : 2018-04-16 DOI: 10.1002/cpmb.59

Barton E. Slatko, Andrew F. Gardner, Frederick M. Ausubel

引用次数: 421

Human Induced Pluripotent Stem Cell Production and Expansion from Blood using a Non-Integrating Viral Reprogramming Vector 利用非整合病毒重编程载体从血液中产生和扩增人诱导多能干细胞

Current Protocols in Molecular Biology Pub Date : 2018-04-16 DOI: 10.1002/cpmb.58

Arun Sharma, Michael Mücke, Christine E. Seidman

引用次数: 10

The Use of the Fluidigm C1 for RNA Expression Analyses of Single Cells Fluidigm C1在单细胞RNA表达分析中的应用

Current Protocols in Molecular Biology Pub Date : 2018-04-16 DOI: 10.1002/cpmb.55

Daniel M. DeLaughter

引用次数: 19

Pyrosequencing: Powerful and Quantitative Sequencing Technology 焦磷酸测序:强大的定量测序技术

Current Protocols in Molecular Biology Pub Date : 2018-02-16 DOI: 10.1002/0471142727.mb0715s104

Martin Kreutz, Norbert Hochstein, Julia Kaiser, Frank Narz, Ralf Peist

引用次数: 10

Helicase-Dependent Amplification of Nucleic Acids 核酸解旋酶依赖性扩增

Current Protocols in Molecular Biology Pub Date : 2018-02-16 DOI: 10.1002/0471142727.mb1511s104

Yun Cao, Hyun-Jin Kim, Ying Li, Huimin Kong, Bertrand Lemieux

{"title":"Helicase-Dependent Amplification of Nucleic Acids","authors":"Yun Cao, Hyun-Jin Kim, Ying Li, Huimin Kong, Bertrand Lemieux","doi":"10.1002/0471142727.mb1511s104","DOIUrl":"10.1002/0471142727.mb1511s104","url":null,"abstract":"Helicase-dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt-end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip. Curr. Protoc. Mol. Biol. 104:15.11.1-15.11.12. © 2013 by John Wiley & Sons, Inc.","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"104 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471142727.mb1511s104","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32101345","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 14

Whole-Genome Amplification of Single-Cell Genomes for Next-Generation Sequencing 单细胞基因组全基因组扩增用于下一代测序

Current Protocols in Molecular Biology Pub Date : 2018-02-16 DOI: 10.1002/0471142727.mb0714s104

Christian Korfhage, Evelyn Fisch, Evelyn Fricke, Silke Baedker, Dirk Loeffert

{"title":"Whole-Genome Amplification of Single-Cell Genomes for Next-Generation Sequencing","authors":"Christian Korfhage, Evelyn Fisch, Evelyn Fricke, Silke Baedker, Dirk Loeffert","doi":"10.1002/0471142727.mb0714s104","DOIUrl":"10.1002/0471142727.mb0714s104","url":null,"abstract":"DNA sequence analysis and genotyping of biological samples using next-generation sequencing (NGS), microarrays, or real-time PCR is often limited by the small amount of sample available. A single cell contains only one to four copies of the genomic DNA, depending on the organism (haploid or diploid organism) and the cell-cycle phase. The DNA content of a single cell ranges from a few femtograms in bacteria to picograms in mammalia. In contrast, a deep analysis of the genome currently requires a few hundred nanograms up to micrograms of genomic DNA for library formation necessary for NGS sequencing or labeling protocols (e.g., microarrays). Consequently, accurate whole-genome amplification (WGA) of single-cell DNA is required for reliable genetic analysis (e.g., NGS) and is particularly important when genomic DNA is limited. The use of single-cell WGA has enabled the analysis of genomic heterogeneity of individual cells (e.g., somatic genomic variation in tumor cells). This unit describes how the genome of single cells can be used for WGA for further genomic studies, such as NGS. Recommendations for isolation of single cells are given and common sources of errors are discussed. Curr. Protoc. Mol. Biol. 104:7.14.1-7.14.11. © 2013 by John Wiley & Sons, Inc.","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"104 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471142727.mb0714s104","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32101346","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 11

Identification of Mutations in Zebrafish Using Next-Generation Sequencing 利用新一代测序技术鉴定斑马鱼的突变

Current Protocols in Molecular Biology Pub Date : 2018-02-16 DOI: 10.1002/0471142727.mb0713s104

Katrin Henke, Margot E. Bowen, Matthew P. Harris

{"title":"Identification of Mutations in Zebrafish Using Next-Generation Sequencing","authors":"Katrin Henke, Margot E. Bowen, Matthew P. Harris","doi":"10.1002/0471142727.mb0713s104","DOIUrl":"10.1002/0471142727.mb0713s104","url":null,"abstract":"Whole-genome sequencing (WGS) has been used in many invertebrate model organisms as an efficient tool for mapping and identification of mutations affecting particular morphological or physiological processes. However, the application of WGS in highly polymorphic, larger genomes of vertebrates has required new experimental and analytical approaches. As a consequence, a wealth of different analytical tools has been developed. As the generation and analysis of data stemming from WGS can be unwieldy and daunting to researchers not accustomed to many common bioinformatic analyses and Unix-based computational tools, we focus on how to manage and analyze next-generation sequencing datasets without an extensive computational infrastructure and in-depth bioinformatic knowledge. Here we describe methods for the analysis of WGS for use in mapping and identification of mutations in the zebrafish. We stress key elements of the experimental design and the analytical approach that allow the use of this method across different sequencing platforms and in different model organisms with annotated genomes. Curr. Protoc. Mol. Biol. 104:7.13.1-7.13.33. © 2013 by John Wiley & Sons, Inc.","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"104 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471142727.mb0713s104","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32101820","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

Construction of Mutant Alleles in Saccharomyces cerevisiae without Cloning: Overview and the Delitto Perfetto Method 非克隆酿酒酵母突变等位基因的构建:综述与Delitto perfect法

Current Protocols in Molecular Biology Pub Date : 2018-02-16 DOI: 10.1002/0471142727.mb1310cs104

Zarmik Moqtaderi, Joseph V. Geisberg

{"title":"Construction of Mutant Alleles in Saccharomyces cerevisiae without Cloning: Overview and the Delitto Perfetto Method","authors":"Zarmik Moqtaderi, Joseph V. Geisberg","doi":"10.1002/0471142727.mb1310cs104","DOIUrl":"10.1002/0471142727.mb1310cs104","url":null,"abstract":"Traditionally, methods for introducing specific new mutations at target loci in the yeast genome have involved the preparation of disruption or gene-replacement cassettes via multiple cloning steps. Sequences used for targeting these cassettes or integrating vectors are typically several hundred base pairs long. A variety of newer methods rely on the design of custom PCR oligonucleotides containing shorter sequence tails (∼50 nt) for targeting the locus of interest. These techniques obviate the need for cloning steps and allow construction of mutagenesis cassettes by PCR amplification. Such cassettes may be used for gene deletion, epitope tagging, or site-specific mutagenesis. The strategies differ in several ways, most notably with respect to whether they allow reuse of the selection marker and whether extra sequences are left behind near the target locus. This unit presents a summary of methods for targeted mutagenesis of Saccharomyces cerevisiae loci without cloning, including PCR-based allele replacement, delitto perfetto, and MIRAGE. Next, a protocol is provided for the delitto perfetto PCR- and oligonucleotide-based mutagenesis method, which offers particular advantages for generating several different mutant alleles of the same gene. Curr. Protoc. Mol. Biol. 104:13.10C.1-13.10C.17. © 2013 by John Wiley & Sons, Inc.","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"104 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471142727.mb1310cs104","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32101344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 12

Using Google Reverse Image Search to Decipher Biological Images 使用谷歌反向图像搜索破译生物图像

Current Protocols in Molecular Biology Pub Date : 2018-02-15 DOI: 10.1002/0471142727.mb1913s111

Jennifer L. Mamrosh, David D. Moore

引用次数: 11

Laser Microdissection–Mediated Isolation and In Vitro Transcriptional Amplification of Plant RNA 激光微解剖介导的植物RNA分离及体外转录扩增

Current Protocols in Molecular Biology Pub Date : 2018-02-15 DOI: 10.1002/0471142727.mb25a03s112

Divya Chandran, Michael J. Scanlon, Kazuhiro Ohtsu, Marja C.P. Timmermans, Patrick S. Schnable, Mary C. Wildermuth

引用次数: 22