|
|
|
|
| LEADER |
00000cam a22000007a 4500 |
| 001 |
SCIDIR_on1407093353 |
| 003 |
OCoLC |
| 005 |
20231120010758.0 |
| 006 |
m o d |
| 007 |
cr |n||||||||| |
| 008 |
231103s2023 mau o 000 0 eng d |
| 040 |
|
|
|a YDX
|b eng
|c YDX
|d SFB
|d OPELS
|d GZM
|d OCLKB
|d OCLCO
|
| 019 |
|
|
|a 1407367585
|
| 020 |
|
|
|a 9780443157714
|q (electronic bk.)
|
| 020 |
|
|
|a 0443157715
|q (electronic bk.)
|
| 020 |
|
|
|z 0443157707
|
| 020 |
|
|
|z 9780443157707
|
| 035 |
|
|
|a (OCoLC)1407093353
|z (OCoLC)1407367585
|
| 050 |
|
4 |
|a QP623.5.C36
|
| 082 |
0 |
4 |
|a 572.88
|2 23/eng/20231109
|
| 245 |
0 |
0 |
|a Enzymes in RNA science and biotechnology.
|n Part A /
|c edited by Ryota Yamagami, Chun Kit Kwok.
|
| 250 |
|
|
|a First edition.
|
| 264 |
|
1 |
|a Cambridge, MA :
|b Academic Press,
|c 2023.
|
| 300 |
|
|
|a 1 online resource (xviii, 281 pages).
|
| 336 |
|
|
|a text
|b txt
|2 rdacontent
|
| 337 |
|
|
|a computer
|b c
|2 rdamedia
|
| 338 |
|
|
|a online resource
|b cr
|2 rdacarrier
|
| 490 |
1 |
|
|a Methods in enzymology ;
|v 691
|
| 505 |
0 |
|
|a Front Cover -- Series Page -- Methods in Enzymology, -- Copyright -- Contents -- Contributors -- Preface -- Section 1: Reverse transcriptase Part I (discovery, preparation, general utilization) -- Chapter One: End-to-end RT-PCR of long RNA and highly structured RNAEnd-to-end RT-PCR of long RNA and highly structured RNA -- 1 Introduction -- 2 General methods of RT-PCR using MarathonRT -- 2.1 Before you begin -- 2.2 Key resources table (Table 2) -- 2.3 Materials and equipment -- 2.4 Step-by-step method details -- 2.4.1 Annealing RT primers to RNA templates -- 2.4.2 Preparing the reverse transcription mixture -- 2.4.3 Carrying out the reverse transcription -- 2.4.4 Programming a thermal cycler -- 2.4.5 Preparing the PCR mixture -- 2.4.6 Carrying out the PCR amplification -- 2.4.7 Examining the PCR products by electrophoresis -- 2.5 Expected outcomes -- 2.6 Advantages -- 2.7 Limitations -- 2.8 Optimization and troubleshooting -- 2.8.1 Potential problems -- 2.8.2 Potential solutions for optimizing the procedure -- 3 Summary -- Acknowledgment -- References -- Chapter Two: RT-based Sanger sequencing of RNAs containing complex RNA repetitive elements -- 1 Introduction -- 2 General methods of RNA Sanger sequencing using MarathonRT -- 2.1 Before you begin -- 2.2 Key resources table (Table 4) -- 2.3 Materials and equipment -- 2.4 Step-by-step method details -- 2.4.1 Annealing RT primers to RNA templates -- 2.4.2 Preparing the reverse transcription mixture -- 2.4.3 Carrying out the reverse transcription -- 2.4.4 Polyacrylamide gel electrophoresis (PAGE) to analyze the RNA sequence -- 2.5 Expected outcomes -- 2.6 Advantages -- 2.7 Limitations -- 2.8 Optimization and troubleshooting -- 2.8.1 Low primer extension efficiency -- 2.8.2 Unreadable sequence -- 3 Summary -- Acknowledgment -- References.
|
| 505 |
8 |
|
|a Chapter Three: Engineering TNA polymerases through iterative cycles of directed evolution -- 1 Introduction -- 2 Library design -- 2.1 Designing a library for homologous recombination -- 2.2 Amplification of the expression vector for Gibson assembly -- 2.3 PCR cleanup and DpnI treatment -- 2.4 Agarose gel purification -- 2.5 Gibson assembly of the parent genes and linear vector and transformation -- 2.6 Fragment generation and homologous recombination -- 2.7 Gibson assembly of the recombination library and linear vector -- 2.8 Plasmid scale-up and purification -- 3 Cell growth, sample preparation for Droplet-based Optical Polymerase Sorting (DrOPS) and plasmid recovery -- 4 Colony picking and activity screen -- 5 Mutagenic PCR -- 6 Notes -- 7 Summary and conclusions -- Acknowledgments -- References -- Section 2: Reverse transcriptase Part II (RNA structure mapping and determination)biosynthesis -- Chapter Four: RNA G-quadruplex (rG4) structure detection using RTS and SHALiPE assays -- 1 Introduction -- 2 Materials -- 2.1 Before you begin -- 2.2 In vitro transcription and RNA purification -- 2.3 Gel formation, running and analysis -- 2.4 RTS -- 2.4.1 Preparation of buffer -- 2.4.2 Reagent and instrument -- 2.5 SHALiPE -- 2.5.1 Preparation of buffer -- 2.5.2 Reagent and instrument -- 2.5.3 RNA purification and quantification -- 3 Methods -- 3.1 In vitro transcription (timing 2 day) -- 3.2 Gel formation (timing 1 h) -- 3.3 RTS assay (timing 40 min) -- 3.4 SHALiPE assay (timing 1.5 h) -- 3.4.1 NAI reaction -- 3.4.2 Reverse transcription (timing 30 min) -- 3.5 Gel running and analysis (timing 2 h) -- 4 Notes -- 5 Summary and conclusion -- Funding -- References -- Chapter Five: Structure-seq of tRNAs and other short RNAs in droplets and in vivo -- 1 Introduction -- 2 Materials -- 2.1 General equipment -- 2.2 General materials -- 3 Methods.
|
| 505 |
8 |
|
|a 3.1 Overview of tRNA Structure-seq -- 3.2 Preparation of reagents -- 3.3 PCR amplification of single-stranded DNA oligopools -- 3.4 BsaI restriction endonuclease treatment -- 3.5 In vitro transcription of RNA using hemi-duplexed DNA -- 3.6 In vitro transcription of RNA using BsaI-cleaved dsDNA oligopools -- 3.7 Purification of in vitro transcribed RNA -- 3.8 Removal of 52 triphosphates for radiolabeling -- 3.9 Radioactive 32P 52-end labeling -- 3.10 32-end fluorescent labeling -- 3.11 Radiation-detected accumulation studies -- 3.12 Fluorescence-detected accumulation studies -- 3.13 Preliminary DMS reaction for tRNAs in droplets -- 3.14 tRNA Structure-seq in droplets (Library preparation) -- 3.15 tRNA Structure-seq in vivo (Library preparation) -- 3.16 tRNA Structure-seq analysis -- 4 Notes -- Funding -- References -- Chapter Six: Capture the in vivo intact RNA structurome by CAP-STRUCTURE-seqCapture the in vivo intact RNA structurome by CAP-STRUCTURE-seq -- 1 Introduction -- 2 Materials -- 2.1 Preparation of plant materials -- 2.2 RNA structure probing conditions optimization for single-hit kinetics -- 2.2.1 Chemical treatment -- 2.2.2 RNA isolation, treatment, and reverse transcription -- 2.2.3 Urea polyacrylamide gel electrophoresis -- 2.3 CAP-STRUCTURE-seq RNA preparation -- 2.4 CAP-STRUCTURE-seq cDNA library construction -- 3 Equipments -- 4 Methods -- 4.1 Overview -- 4.2 Plant materials and growth conditions -- 4.3 Gel-based 18S rRNA structure probing -- 4.3.1 Synthesis 2-methylnicotinic acid imidazolide (NAI) -- 4.3.2 Optimize the chemical treatment conditions for single-hit kinetics -- 4.3.3 RNA isolation -- 4.3.4 DNase treatment -- 4.3.5 First-strand cDNA synthesis with specific primer -- 4.3.6 Gel analysis of 18S rRNA structure and quantification -- 4.4 (+) SHAPE and () SHAPE CAP-STRUCTURE-seq RNA preparation -- 4.4.1 52cap enrichment.
|
| 505 |
8 |
|
|a 4.4.2 Poly (A) selection -- 4.5 (+) SHAPE and () SHAPE CAP-STRUCTURE-seq cDNA library construction -- 4.5.1 First strand cDNA preparation via reverse transcription -- 4.5.2 Single strand DNA ligation -- 4.5.3 cDNA size selection -- 4.5.4 PCR cycle optimization and PCR pool amplification -- 4.5.5 Size selection of PCR pool amplification products -- 4.6 Library validation -- 4.7 CAP-STRUCTURE-seq analysis -- 4.7.1 Deep sequencing reads mapping -- 4.7.2 SHAPE reactivity calculation -- 5 Notes -- 6 Concluding remarks and perspective -- Acknowledgments -- Conflict of interest -- References -- Chapter Seven: Sequencing-based analysis of RNA structures in living cells with 2A3 via SHAPE-MaPSequencing-based analysis of RNA structures in living cells with 2A3 via SHAPE-MaP -- 1 Introduction -- 2 Materials -- 2.1 Equipment -- 2.2 Oligonucleotides and adapters -- 2.2.1 Barcodes -- 2.3 Reagents and kits -- 2.4 Buffers -- 2.4.1 Synthesis of 2-aminopyridine-3-carboxylic acid imidazolide (2A3) -- 2.4.2 Preparation of 2X T4 RNA Ligase Buffer -- 3 SHAPE probing -- 3.1 Overview -- 3.2 In-cell probing of Escherichia coli cells -- 3.3 RNA extraction -- 3.3.1 RNA quality control -- 4 Library preparation -- 4.1 Overview -- 4.2 RNA fragmentation -- 4.3 Traditional method -- 4.3.1 Overview -- 4.3.2 End-repair of RNA fragments -- 4.3.3 52 and 32 adapter ligation -- 4.4 Fast-forward method -- 4.4.1 Overview -- 4.4.2 End-repair of RNA fragments -- 4.4.3 52 adapter ligation -- 4.5 Reverse transcription -- 4.6 Library enrichment -- 4.7 Library quantification and QC -- 5 Data analysis -- 5.1 Overview -- 5.2 Reference index preparation -- 5.3 Data preprocessing and read mapping -- 5.4 Mutation counting -- 5.5 Reactivity calculation -- 5.6 Structure prediction -- 5.6.1 Optimization of folding parameters -- 6 Notes -- 7 Summary and conclusions -- Funding -- References.
|
| 505 |
8 |
|
|a Section 3: RNA polymerase (discovery, preparation, development/engineering and application) -- Chapter Eight: Making RNA: Using T7 RNA polymerase to produce high yields of RNA from DNA templatesMaking RNA -- 1 Introduction -- 2 DNA template design for in vitro transcription -- 2.1 General design principle -- 2.2 Linearized plasmid DNA as template -- 2.3 PCR-amplified DNA as template -- 3 General methods of in vitro transcription using T7 RNA polymerase -- 3.1 Before you begin -- 3.2 Key resources table -- 3.3 Materials and equipment -- 3.4 Step-by-step method details -- 3.4.1 Assembling the in vitro transcription reaction mixture -- 3.4.2 In vitro transcription -- 3.4.3 Optional steps to increase in vitro transcription efficiency -- 3.4.4 After in vitro transcription -- 3.5 Expected outcomes -- 3.6 Advantages -- 3.7 Optimization and troubleshooting -- 3.7.1 General troubleshooting tips -- 3.7.2 Low transcription yield or failed transcription -- 3.7.3 Degradation/truncation of RNA product -- 3.7.4 Aggregation of RNA product -- 4 Preparation of specialized RNA transcripts -- 4.1 Preparation of radioactively labeled (body-labeled) RNA transcripts -- 4.1.1 Additional reagents, buffers and materials -- 4.1.2 Radioactive labeling reaction setup and procedure -- 4.1.3 Notes -- 4.2 Preparation of catalytic RNA molecules -- 4.3 Preparation of long RNA molecules -- 5 Purification workflow for efficient recovery of high-purity RNA transcripts -- 5.1 PAGE gel electrophoresis-based denaturing purification -- 5.1.1 Equipment -- 5.1.2 Reagent and buffers -- 5.1.3 Procedures for denaturing PAGE gel purification -- 5.2 Size exclusion column-based native purification -- 5.2.1 Equipment -- 5.2.2 Reagent and buffers -- 5.2.3 Procedures of SEC purification -- 6 Summary -- Acknowledgments -- References.
|
| 650 |
|
0 |
|a Catalytic RNA.
|
| 650 |
|
0 |
|a Enzymes
|x Biotechnology.
|
| 650 |
|
6 |
|a Ribozymes.
|0 (CaQQLa)201-0224271
|
| 650 |
|
6 |
|a Enzymes
|0 (CaQQLa)201-0002557
|x Biotechnologie.
|0 (CaQQLa)201-0378829
|
| 776 |
0 |
8 |
|i ebook version :
|z 9780443157714
|
| 776 |
0 |
8 |
|c Original
|z 0443157707
|z 9780443157707
|w (OCoLC)1370927301
|
| 830 |
|
0 |
|a Methods in enzymology ;
|v 691.
|
| 856 |
4 |
0 |
|u https://sciencedirect.uam.elogim.com/science/bookseries/00766879/691
|z Texto completo
|
