The handbook of plant functional genomics: concepts and protocols
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| Format: | Book |
|---|---|
| Language: | English |
| Published: |
Weinheim
WILEY-VCH
2008
|
| Series: | Molecular plant biology handbook series
|
| Subjects: | |
| Online Access: | Inhaltstext Inhaltsverzeichnis |
| Physical Description: | XXVIII, 548 S. Ill., graph. Darst. |
| ISBN: | 9783527318858 3527318852 |
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| 020 | |a 3527318852 |c Gb. : ca. EUR 159.00 (freier Pr.), ca. sfr 251.00 (freier Pr.) |9 3-527-31885-2 | ||
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| 245 | 1 | 0 | |a The handbook of plant functional genomics |b concepts and protocols |c ed. by Günter Kahl ... |
| 264 | 1 | |a Weinheim |b WILEY-VCH |c 2008 | |
| 300 | |a XXVIII, 548 S. |b Ill., graph. Darst. | ||
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| 490 | 0 | |a Molecular plant biology handbook series | |
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VII
Contents
Preface
XIX
List of Contributors
XXI
I Transcriptome Analysis
1
A Whole Genome Expression Analysis
1
1
Single Cell Expression Profiling: Transcript and Protein Analyses
in Isolated Higher Plant Gametes and
Zygotes
3
Stefan
Scholten
and
Erhard Kranz
1.1
Introduction
3
1.2
Microdissection,
Cell Isolation
6
1.3
In Vitro Fertilization
6
1.4
Techniques for Molecular Analyses of Single Cell Types
7
1.4.1
Sampling of Single, Living Cells
7
1.4.2
Analyses of Gene Expression
8
1.4.2.1
Single Cell Gene-by-Gene Analysis
8
1.4.2.2
Amplification of Whole cDNA Populations
9
1.4.2.3
Quantification of Transcript Levels
11
1.4.2.4
Library Construction and
EST
Sequencing
12
1.4.2.5
Targeted Approaches Using cDNA Subtraction
12
1.4.2.6
Microarray Analyses
13
1.5
Analyses of Protein Expression
14
1.6
Prospects
15
References
16
2
AFLP-Based
RNA
Fingerprinting: Novel Variants and Applications
21
Christian
W.B. Bachern, Wim
H.
Vriezen, Craita E.
Bita, and
Asun
Fernandez del Carmen
2.1
Introduction
21
2.2
Methods and Protocols
23
VIII Contents
2.2.1
Theoretical Considerations
23
2.2.2
State-of-the-Art cDNA-AFLP Protocol
24
2.2.2.1
Isolation of cDNA Fragments
24
2.2.2.2
Non-Selective Pre-Amplification
25
2.2.2.3
Selective Amplification-Reaction Using 33P-Labeled Primer and
Gel Analysis
26
2.2.2.4
Downstream Analysis
29
2.3
Applications of the Technology
31
2.3.1
Fruit Development
31
2.3.2
Tuber Development
32
2.3.3
Transcript BSA
32
2.3.4
Domain Profiling
33
2.3.5
VIDISCA
33
2.4
Perspectives
34
References
34
3
SuperSACE: The Most Advanced
Transcriptome
Technology for Functional Genomics
37
Ryohei Terauchi, Hideo Matsumura,
Detlev
H.
Krüger,
and
Günter Kahl
3.1
Introduction
37
3.2
Methods and Protocols
40
3.2.1
Linker Preparation
41
3.2.2
RNA
Sample
42
3.2.3
cDNA Synthesis
43
3.2.4
Tag Extraction from cDNA
43
3.2.5
Purification of linker-Tag Fragment
44
3.2.6
Ditag Formation and Amplification
44
3.2.7
Tag Extraction from Sequence Data
45
3.3
Applications of the Technology
47
3.3.1
Interaction
Transcriptome
47
3.3.2
Application of SuperSAGE to Non-Model Organisms
47
3.3.3
SuperSAGE-Array
48
3.3.4
GMAT
49
3.4
Perspectives
49
References
51
4
From
CACE
to DeepCACE: High-Throughput Transcription Start Site
and Promoter
Identification
for
Cene
Network Analysis
55
Matthias Harbers, Thomas Werner, and
Piero Cominci
4.1
From Genomes to
Transcriptomes
55
4.2
Addressing the Complexity of
Transcriptomes
56
4.3
The Shift From CAGE to DeepCAGE
57
4.4
Applications of CAGE and DeepCAGE Libraries
58
4.5
Preparation of a DeepCAGE Library
59
4.6
CAGE Data Analysis and Genome Mapping Approaches
62
Contents IX
4.7
Expression Profiling: Putting CAGE Tags into a Biological
Context
67
4.8
Perspectives
68
References
70
5
Gene Identification Signature-Paired End diTagging (CIS-PET):
A Technology for
Transcriptome
Characterization
77
Patrick Ng, Yen-Ling Lee, Chia-Lin Wei, and Yijun Ruan
5.1
Introduction
77
5.1.1
Microarray Analysis
79
5.1.2
cDNA Sequencing, Including
EST-
and flcDNA-Sequencing
79
5.1.3
DNA-Tagging Methods
80
5.1.4
Advanced
DNA
Sequencing Technologies
81
5.2
Protocol
82
5.2.1
Construction of a GIS-PET flcDNA Library
84
5.2.1.1
Reverse-Transcription of mRNA (polyA-RNA) Sample
84
5.2.1.2
Oxidation
86
5.2.1.3
BiotinylationofRNAEnds
86
5.2.1.4
RNaseONE Selection for Full-Length
(-)
cDNA/RNA
Heteroduplex
86
5.2.1.5
Binding Biotinylated
(-)
cDNA/RNA Heteroduplex
to Streptavidin Beads
87
5.2.1.6
Alkaline Hydrolysis to Release
(-)
Strand flcDNA
88
5.2.1.7
Double-Stranded cDNA (ds cDNA) Synthesis
88
5.2.1.8
Further Processing of ds flcDNA
89
5.2.1.9
cDNA Size Fractionation
90
5.2.1.10
Cloning of flcDNA in pGIS4a Vector
90
5.2.1.11
Perform QC on flcDNA Library
92
5.2.2
Construction of a Single-PET Library
92
5.2.2.1
Plasmid
DNA
Preparation
92
5.2.2.2
Tagging by
Mme
I Digestion
93
5.2.2.3
Intramolecular Circularization to Create Single-PET
Plasmids
93
5.2.2.4
Transform Cells
94
5.2.2.5
Perform QC on
GIS Single-PET
Library
94
5.2.3
Construction of a GIS-PET Sequencing Library for
Sänger
Sequencing of Ditags
95
5.2.3.1
Single-PET Plasmid
DNA
Preparation
95
5.2.3.2
Bam HI-Digestion to Release Single-PETs
95
5.2.3.3
PAGE-Purifkation of 50-bp BamHI-Cohesive Single PETs
96
5.2.3.4
PET Concatenation
97
5.2.3.5
Purification of Concatenated PETs
97
5.2.3.6
Cloning Concatenated PETs in pZErO-1 Vector
98
5.2.3.7
Transform Cells
99
5.2.3.8
Carry out QC on GIS-PET Sequencing Library
99
X
Contents
5.2.4
ConstractìonofdiPETsfor454-Sequencing
200
5.2.4.1
Single-PET Plasmid
DNA
Preparation
100
5.2.4.2
Bse
RI
Linearization of Single-PET Plasmid
DNA 100
5.2.4.3
BamHI Digestion to Release Asymmetric PETs
101
5.2.4.4
Recovery and Quantitation of Purified Asymmetric PETs
101
5.2.4.5
Formation of diPETs
102
5.3
Data Analysis
103
5.4
Discussion
103
5.5
Perspectives
105
References
109
6
High-Throughput Functional Screening of Genes In
Planta
113
Thomas
Berberich,
Yoshihiro Takahashi, Hiromasa Saitoh, and
Ryohei Terauchi
6.1
Introduction
113
6.2
Methods and Protocols
115
6.2.1
Extraction of Total
RNA
116
6.2.2
Purification of Poly(A)+-mRNA
119
6.2.3
Synthesis of cDNA and Ligation to Binary, PVX-Based
Expression Vectors
121
6.2.4
Amplification of the cDNA Library in
E. coli
124
6.2.5
Transformation of cDNA library into Agrobacterium
tumefaciens Cells
126
6.2.5.1
Preparation of Competent Agrobacterium tumefaciens
Cells
326
6.2.6
Toothpick Inoculation of Leaves
128
6.2.7
Agroinfiltration
129
6.2.8
Recovery of the cDNA Fragments
130
6.3
Application of the Technology
131
6.4
Perspectives
133
References
134
7
Microarrays as Tools to Decipher
Transcriptomes
in Symbiotic
Interactions
137
Helge Küster
and
Anke
Becker
7.1
Introduction
137
7.2
Methods and Protocols
143
7.2.1
Spotting and Storage of
MtlĎkOLIl/MtlókOLIlPlus
70mer
Oligonucleotide Microarrays
143
7'.2.2
Synthesis of Targets by Indirect Reverse Transcription
Cy-Labeling
144
7.2.2.1
Components Stored at
-20 °С 144
7.2.2.2
Components Stored at
4
JC
(-20 °С
after Aliquoting
in
1/10
Volumes)
145
7.2.2.3
Components Stored at Room Temperature
145
Contents
XI
7.2.2.4 Reverse Transcription
of
Total
RNA
to obtain
Aminoalłyl-
Labeled
First-Strand cDNA
145
7.2.2.5
Hydrolysis of
RNA
246
7.2.2.6
Clean-Up of
Aminoalłyl-
Labeled First-Strand cDNA
146
7.2.2.7
Coupling of Fluorescent Dyes to Aminoallyl-Labeled
First-Strand cDNA
147
7.2.2.8
Quenching of all Remaining NHS Esters
147
7.2.2.9
Clean-up of Fluorescently Labeled Targets
147
7.2.2.10
Quality Control of Fluorescently Labeled Targets
148
7.2.3
Pre-Processing, Hybridization and Scanning of
MtlókOLIl/
MtlókOLIlPlus
Microarrays
148
7.2.4
Handling and Evaluation of Microarray Data
150
7.3
Applications of the Technology
151
7.3.1
Microarray-Based Identification of Medicago truncatula Genes
Induced during Different
Arbuscular Mycorrhizal
Interactions
151
7.3.2
Microarray-Based Identification of Medicago truncatula Genes
Activated during Nodulation and Mycorrhization
254
7.4
Perspectives
156
References
157
В
Gene-by-Cene Analysis
163
8
Genome-Wide Analysis of mRNA Expression by Fluorescent
Differential Display
165
Suping Zhou, Jonathan D. Meade, Samuel Nahashon, Blake R. Shester,
Jamie C.
Walden,
Then Cuo, Julia Z. Liang, Joshua G. Liang, and Peng Liang
8.1
Introduction
165
8.2
Methods and Protocols
169
8.2.1
Materials
169
8.2.1.1
Total
RNA
Isolation and Removal of Genomic
DNA
from
Total
RNA
169
8.2.1.2
Single-Strand cDNA Synthesis by Reverse Transcription
170
8.2.1.3
Fluorescent Differential Display-PCR (FDD-PCR)
270
8.2.1.4
Gel Electrophoresis
270
8.2.1.5
Reamplification
of Selected Differentially Expressed Bands
171
8.2.1.6
Cloning of Reamplified PCR Products
171
8.2.1.7
Verification of Cloned PCR Products
271
8.2.1.8
Confirmation of Differential Gene Expression by Northern Blot
172
8.2.2
Methods
172
8.2.2.1
Total
RNA
Isolation and Removal of Genomic
DNA 172
8.2.2.2
Gel Preparation
274
8.2.2.3
RNA
Loading Sample Preparation
274
8.2.2.4
Single-Strand cDNA Synthesis by Reverse Transcription
174
8.2.2.5
Fluorescent Differential Display-PCR
175
XII Contents
8.2.2.6
Gel Electrophoresis
176
8.2.2.7
Reamplification
of Selected Differentially Expressed
cDNA Bands
177
8.2.2.8
Cloning of Reamplified PCR Products
179
8.2.2.9
Verification of the Cloned Inserts
180
8.2.2.10
Confirmation of Differential Gene Expression by Northern Blot
181
8.3
Applications of the Technology
183
8.4
Perspectives
284
References
285
9
Real-Time Quantitation of MicroRNAs by TaqMan
MicroRNA Assays
287
Toni
L
Ceccardi,
Marianna M.
Goldrick, Peifeng Ren, Rick
С
Conrad, and
Caifu
Chen
9.1
Introduction
187
9.1.1
What are microRNAs?
287
9.1.2
Why are Researchers Interested?
288
9.1.3
Current Technologies for miRNA Quantitation
289
9.2
Methods and Protocols
290
9.2.1
Bioinformatic Tools for miRNA Discovery
290
9.2.2
MicroRNA Isolation from Plants
292
9.2.2.1
Extraction from Plant Tissue
192
9.2.2.2
PCR Directly from Cells in Culture
293
9.2.3
Description of TaqMan MicroRNA Assays
194
9.2.3.1
Principle of TaqMan MicroRNA Assays
194
9.2.3.2
Performing the TaqMan MicroRNA Assay
295
9.2.4
Data Normalization
296
9.3
Applications of the Technology
297
9.3.1
Quantitation of miRNAs
197
9.3.2
Absolute Quantitation of miRNAs
298
9.3.3
Expression Profiling of miRNAs
298
9.3.4
Verification of Predicted Novel miRNAs
299
9.3.5
MicroRNAs in Plant Growth and Development
299
9.3.6
Discovery of miRNA
Biomarkers
202
9.3.7
Discovery and Validation of Plant miRNA Targets
202
9.4
Perspectives
202
References
203
II Gene Silencing, Mutation Analysis and Functional Genomics
207
10
RNA
Interference
209
Chris A. Brosnan, Emily J. McCallum,
José
R.
Botella,
and Bernard
J.
Carroll
10.1
Introduction
209
10.2
Methods and Protocols
213
Contents XIII
10.3
Applications of the Technology
216
10.3.1
Targeting
Transgenes
in Arabidopsis using RNAi
216
10.3.2
Tissue-Specific
RNA
Silencing of an Endogenous Gene
in Tobacco
218
10.3.3
Advantages of Using RNAi in Plant Functional
Genomics
219
10.4
Perspectives
219
References
220
ΊΊ
Extending Functional Cenomics: VICS for Model and Crop Plants
227
Steven Bernacki, John Richard Tuttle, Nooduan Muangsan, and
Dominique Robertson
11.1
Introduction
227
11.2
Methods and Protocols
232
11.2.1
Constructing Geminiviras VIGS Vectors
232
11.2.1.1
Structure of Geminiviras Plasmids
233
11.2.1.2
Construction of an
ARI
Replacement Vector
234
11.2.1.3
Construction of an Insertion Vector
235
11.2.2
Silencing an Endogenous Gene
236
11.2.2.1
Visible Markers for Testing and Optimizing VIGS
236
11.2.2.2
Cloning a Target Gene Fragment(s) into the CaLCuVAiChll
Vector
237
11.2.2.3
Plant Preparation
237
11.2.2.4
Microprojectile Bombardment
238
11.2.2.5
Assessment of VIGS
239
11.3
Applications of the Technology
241
11.4
Perspectives
242
References
243
12
TILLING: A Reverse Genetics and a Functional Genomics Tool
in Soybean
251
Khalid
Mekšem,
Shiming Liu, Xiao Hong Liu, Azizjamai, Melissa Coellner
Mitchum, Abdelhafid Bendahmane, and Tarik El-Mellouki
12.1
Introduction
251
12.2
Methods and Protocols
252
12.2.1
Production of Suitable Mutant Population for TILLING
252
12.2.2 DNA
Extraction and
DNA
Construction of Pools
253
12.2.3
TILLING Screening for Mutations
254
12.2.3.1
Gene-Specific Primers for TILLING
254
12.2.3.2
PCR Amplification and Heteroduplex Formation
255
12.2.3.3
МІЗ
-Tailed
PCR Amplification for TILLING
255
12.2.3.4
Endonuclease Digestion and Purification of the Amplified
DNA 256
12.2.3.5
Gel Electrophoresis and Image Analysis
257
12.3
Applications of TILLING to Soybean
258
XIV Contents
12.3.1 Mutation Discovery,
Density
and Distribution in
two Mutagenized
Soybean Populations
258
12.3.2
Confirmation and Segregation Patterns of TILLING Mutations
in Soybean
260
12.4
Discussion and Perspectives
262
References
264
13
Transposon Tagging in Cereal Crops
267
Liza]. Conrad, Kazuhiro Kikuchi, and Thomas P. Brutnell
13.1
Insertional Mutagenesis in Plants
268
13.2
Transposon Tagging in Maize
270
13.2.1
Mutator Insertional Mutagenesis
270
13.2.2
Activator/Dissociation Mutagenesis
274
13.2.2.1
Forward Genetics
274
13.2.2.2
Reverse Genetics in Maize Using Ds
275
13.3
Large-Scale Reverse Genetics in Rice
275
13.3.1
Tosi
7
in Rice
276
13.3.2
The Maize
Ас
/Ds
Transposons
in Rice
277
13.3.3
En/Spm
279
13.4
Ас
/Ds
Transposon Tagging in Barley
280
13.5
Future Direction of Tagging in Cereals
281
13.5.1
Potential for an Endogenous Candystripel Tagging System
in Sorghum
281
13.5.2
Transposon-Mediated Deletions in Maize
282
13.5.3
Future Tagging Resources in Rice
282
13.5.4
Saturation Mutagenesis
282
References
282
14
Fast Neutron Mutagenesis for Functional Cenomics
291
Christian Rogers and
Сі
/es
Oldroyd
14.1
Introduction
291
14.1.1
Advantages of Fast Neutron Mutagenesis
291
14.1.2
Features of Fast Neutron Mutagenesis
292
14.1.3
Fast Neutron Mutagenesis for Reverse Genetics
294
14.2
Methods and Protocols
294
14.2.1
Screening Strategies
294
14.2.2
Automation
297
14.2.3
Establishing the Populations
298
14.2.4
Pooling Strategies
299
14.2.5
Characterization of the Populations
301
14.3
Applications of the Technology
301
14.3.1
Targeting Small Genes
302
14.3.2
Deletions Can Span Multiple Genes
303
14.3.3
Fast Neutron Reverse Genetics for Crop Improvement
303
Contents XV
14.4
Perspectives
304
References
304
III Computational Analysis
307
15
Bioinformatics Tools to Discover Co-Expressed
Cenes
in Plants
309
Yoshiyuki Ogata, Nozomu Sakurai, Nicholas J.
Provali,
Dirk Steinhauser,
and Leonard
Krall
15.1
Introduction
309
15.2
The Expression Angler Tool of the Botany Array Resource
311
15.2.1
Methods and Protocols
311
15.2.2
Applications of the Technology
317
15.2.3
Perspectives
319
15.3
The CSB.DB Tool
320
15.3.1
Introduction
320
15.3.2
Methods and Protocols
321
15.3.2.1
Simple Protocol for the Use of CSB.DB
322
15.4
The KaPPA-View
2:
Co-Expression Analysis on the Plant Metabolic
Pathway Maps
324
15.4.1
Introduction
324
15.4.2
Application of the Technology
324
15.4.3
Methods and Protocols
326
15.4.4
Perspectives
328
15.5
The KAGIANATool for Co-Expression Network Analysis
of Arabidopsis Genes
328
15.5.1
Introduction
328
15.5.2
Methods and Protocols
329
15.5.2.1
Initial Setting
329
15.5.2.2
Retrieval of Co-Expressed Genes
329
15.5.2.3
The Other Tools of KAGIANA
330
15.5.3
Perspective
331
References
331
16
AthaMap, a Database for the Identification and Analysis of Transcription
Factor Binding Sites in the Arabidopsis thaliana Genome
337
Reinhard Hehl
16.1
Introduction
337
16.2
Methods and Applications
339
16.2.1
Using the Web Interface at http://www.athamap.de/
339
16.2.1.1
The Search Function
339
16.2.1.2
Co-Localization Analysis
342
16.2.1.3
Gene Analysis
343
16.2.1.4
External links
344
References
345
XVI Contents
17
Structural
Phylogenomic Inference of Plant Gene Function
347
Nandini Krishnamurthy, Jim Leebens-Mack, and
Kimmen
Sjölander
17.1
Introduction
347
17.2
Challenges in Protein Function Prediction
349
17.2.1
Gene Duplication
350
17.2.2
Domain Shuffling
350
17.2.3
Speciation
352
17.2
A Propagation of Existing Annotation Errors
352
17.3
The Nomenclature of Homology
352
17.4
Structural Phylogenomic Inference of Function
354
17.5
Recommended Protocols for a Structural Phylogenomic Pipeline
356
17.5.1
Step
1: Homolog
Selection
357
17.5.2
Step
2:
Constructing and Analyzing a Multiple Sequence
Alignment
358
17.5.3
Step
3:
Constructing and Analyzing a Phylogenetic Tree
359
17.5.4
Step
4:
Predicting Function using a Phylogenetic Tree
361
17.6
Web Servers and Databases useful in Phylogenomic Inference
362
17.7
Discussion
363
References
364
18
Structural, Functional, and Comparative Annotation of Plant
Genomes
373
Françoise
Thibaud-Nissen, Jennifer Wortman,
С
Robin Buell, and Wei Zhu
18.1
Introduction
373
18.2
Methods, Protocols, and Applications
374
18.2.1
Structural Annotation
374
18.2.1.1
Cognate Transcript Sequences are the Most Reliable Data Available
378
18.2.1.2 Ab
initio Gene Finders are Good
ORF
Finders
379
18.2.1.3
Integrated Approaches are Ideal Solutions for the Automated
Gene Prediction
379
18.2.1.4
Gene Prediction is an Iterative Process
380
18.2.1.5
Manual duration is still an Indispensable Process
in Gene Prediction
380
18.2.1.6
Other Considerations for Gene Prediction
380
18.2.2
Functional Annotation
381
18.2.2.1
Sequence Similarity
381
18.2.2.2
Domain Searches
382
18.2.2.3
Phylogenomics
382
18.2.2.4
Expression Data
383
18.2.3
Comparative Annotation
383
18.2.3.1
Comparative Annotation Using Transcripts
383
18.2.3.2
Comparative Genomics Using Genome Sequences
385
18.2.3.3
Algorithms for Comparative Genomics
386
18.3
Perspectives
387
References
389
Contents
XVII
19
Large-Scale Cenomic Sequence Comparison and
Cene
Identification
with ClustDB
397
Jürgen
Kleffe
19.1
Introduction
397
19.2
Methods and Protocols
401
19.2.1
Reading Sequences
401
19.2.2
Substring Clusters
402
19.2.3
Maximally Extended Pairs of Common Substrings
405
19.2.4
Match Extension with Errors
406
19.2.5
Complete Matches
407
19.2.6
Reference Query Problems
407
19.2.7
Complementary Sequences
408
19.2.8
Handling Ambiguity Letter Codes
408
19.2.9
Sequence Clusters
409
19.2.10
Memory Analysis
410
19.3
Applications
410
19.3.1
Deriving Clusters of Identical Plant ESTs
411
19.3.2
Deriving Substring Clusters for All Plant ESTs
412
19.3.3
Checking the TIGR Medicago
ВАС
Assembly
412
19.4
Perspectives
413
References
415
IV Functional Cenomics and Emerging Technologies
417
20
Nanotechnologies and Fluorescent Proteins
fòr
in
pianta
Functional
Cenomics
419
C. Neal Stewart Jr.
20.1
Introduction
419
20.2
Green Fluorescent Protein
420
20.3
Protocol: Seeing GFP in Transgenic Plants
424
20.4
Nanotechnology for Monitoring Gene Expression
425
20.4.1
Aptamers and Quantum Dots
425
20.4.2
Molecular Beacons
425
20.4.3
Split GFP Tagging and Detection
426
20.5
Barriers to Implementation
427
20.6
Conclusions
427
References
428
21
New Frontiers in Plant Functional Cenomics Using Next
Generation Sequencing Technologies
431
Robert
С
Nutter
21.1
Introduction
433
21.1.1
Advent of Massively Parallel Sequencing Systems
431
21.1.2
Overview of the Sequencing by Synthesis System
432
XVIII
Contents
21.1.3
Overview of Single Base Extension System
431
21.1.4
Overview of the (SOliD) System
433
21.2
library Generation
434
21.3
Emulsion PCR
436
21.3.1
Bead Purification
436
21.3.2
Bead Deposition
437
21.4
Sequencing by Iigation
437
21.5
Base Calling
439
21.6
Potential Applications
440
21.6.1
Resequencing
440
21.6.2
De novo
Sequencing
440
21.6.3
Gene Expression via Sequence Tags
441
21.6.4
Other Tag-Based Applications
443
21.7
Conclusions
444
References
444
22 454
Sequencing: The Next Generation Tool for Functional
Cenomics
447
Lei Dia, Jan Frederik
Simons, Maithreyan Srinivasan, Thomas Jarvie,
Bruce Taillon, and Michael Egholm
22.1
Introduction
448
22.2
Methods and Protocols
450
22.2.1 DNA
Library Preparation
450
22.2.1.1
Option A: Nebulized library Procedure
451
22.2.1.2
Option B: Amplicon library Procedure
454
22.2.2
Emulsion PCR
456
22.2.3
Loading of PTP and Instrument Run
460
22.2.4
Data Analysis
467
22.2.4.1
Whole Genome Assembly
467
22.2.4.2
Resequencing and Mutation Detection
469
22.2.4.3
Ultra-deep Sequencing
469
22.3
Applications of the Technology
470
References
472
Glossary
477
Index
537 |
| adam_txt |
VII
Contents
Preface
XIX
List of Contributors
XXI
I Transcriptome Analysis
1
A Whole Genome Expression Analysis
1
1
Single Cell Expression Profiling: Transcript and Protein Analyses
in Isolated Higher Plant Gametes and
Zygotes
3
Stefan
Scholten
and
Erhard Kranz
1.1
Introduction
3
1.2
Microdissection,
Cell Isolation
6
1.3
In Vitro Fertilization
6
1.4
Techniques for Molecular Analyses of Single Cell Types
7
1.4.1
Sampling of Single, Living Cells
7
1.4.2
Analyses of Gene Expression
8
1.4.2.1
Single Cell Gene-by-Gene Analysis
8
1.4.2.2
Amplification of Whole cDNA Populations
9
1.4.2.3
Quantification of Transcript Levels
11
1.4.2.4
Library Construction and
EST
Sequencing
12
1.4.2.5
Targeted Approaches Using cDNA Subtraction
12
1.4.2.6
Microarray Analyses
13
1.5
Analyses of Protein Expression
14
1.6
Prospects
15
References
16
2
AFLP-Based
RNA
Fingerprinting: Novel Variants and Applications
21
Christian
W.B. Bachern, Wim
H.
Vriezen, Craita E.
Bita, and
Asun
Fernandez del Carmen
2.1
Introduction
21
2.2
Methods and Protocols
23
VIII Contents
2.2.1
Theoretical Considerations
23
2.2.2
State-of-the-Art cDNA-AFLP Protocol
24
2.2.2.1
Isolation of cDNA Fragments
24
2.2.2.2
Non-Selective Pre-Amplification
25
2.2.2.3
Selective Amplification-Reaction Using 33P-Labeled Primer and
Gel Analysis
26
2.2.2.4
Downstream Analysis
29
2.3
Applications of the Technology
31
2.3.1
Fruit Development
31
2.3.2
Tuber Development
32
2.3.3
Transcript BSA
32
2.3.4
Domain Profiling
33
2.3.5
VIDISCA
33
2.4
Perspectives
34
References
34
3
SuperSACE: The Most Advanced
Transcriptome
Technology for Functional Genomics
37
Ryohei Terauchi, Hideo Matsumura,
Detlev
H.
Krüger,
and
Günter Kahl
3.1
Introduction
37
3.2
Methods and Protocols
40
3.2.1
Linker Preparation
41
3.2.2
RNA
Sample
42
3.2.3
cDNA Synthesis
43
3.2.4
Tag Extraction from cDNA
43
3.2.5
Purification of linker-Tag Fragment
44
3.2.6
Ditag Formation and Amplification
44
3.2.7
Tag Extraction from Sequence Data
45
3.3
Applications of the Technology
47
3.3.1
Interaction
Transcriptome
47
3.3.2
Application of SuperSAGE to Non-Model Organisms
47
3.3.3
SuperSAGE-Array
48
3.3.4
GMAT
49
3.4
Perspectives
49
References
51
4
From
CACE
to DeepCACE: High-Throughput Transcription Start Site
and Promoter
Identification
for
Cene
Network Analysis
55
Matthias Harbers, Thomas Werner, and
Piero Cominci
4.1
From Genomes to
Transcriptomes
55
4.2
Addressing the Complexity of
Transcriptomes
56
4.3
The Shift From CAGE to DeepCAGE
57
4.4
Applications of CAGE and DeepCAGE Libraries
58
4.5
Preparation of a DeepCAGE Library
59
4.6
CAGE Data Analysis and Genome Mapping Approaches
62
Contents IX
4.7
Expression Profiling: Putting CAGE Tags into a Biological
Context
67
4.8
Perspectives
68
References
70
5
Gene Identification Signature-Paired End diTagging (CIS-PET):
A Technology for
Transcriptome
Characterization
77
Patrick Ng, Yen-Ling Lee, Chia-Lin Wei, and Yijun Ruan
5.1
Introduction
77
5.1.1
Microarray Analysis
79
5.1.2
cDNA Sequencing, Including
EST-
and flcDNA-Sequencing
79
5.1.3
DNA-Tagging Methods
80
5.1.4
Advanced
DNA
Sequencing Technologies
81
5.2
Protocol
82
5.2.1
Construction of a GIS-PET flcDNA Library
84
5.2.1.1
Reverse-Transcription of mRNA (polyA-RNA) Sample
84
5.2.1.2
Oxidation
86
5.2.1.3
BiotinylationofRNAEnds
86
5.2.1.4
RNaseONE Selection for Full-Length
(-)
cDNA/RNA
Heteroduplex
86
5.2.1.5
Binding Biotinylated
(-)
cDNA/RNA Heteroduplex
to Streptavidin Beads
87
5.2.1.6
Alkaline Hydrolysis to Release
(-)
Strand flcDNA
88
5.2.1.7
Double-Stranded cDNA (ds cDNA) Synthesis
88
5.2.1.8
Further Processing of ds flcDNA
89
5.2.1.9
cDNA Size Fractionation
90
5.2.1.10
Cloning of flcDNA in pGIS4a Vector
90
5.2.1.11
Perform QC on flcDNA Library
92
5.2.2
Construction of a Single-PET Library
92
5.2.2.1
Plasmid
DNA
Preparation
92
5.2.2.2
Tagging by
Mme
I Digestion
93
5.2.2.3
Intramolecular Circularization to Create Single-PET
Plasmids
93
5.2.2.4
Transform Cells
94
5.2.2.5
Perform QC on
GIS Single-PET
Library
94
5.2.3
Construction of a GIS-PET Sequencing Library for
Sänger
Sequencing of Ditags
95
5.2.3.1
Single-PET Plasmid
DNA
Preparation
95
5.2.3.2
Bam HI-Digestion to Release Single-PETs
95
5.2.3.3
PAGE-Purifkation of 50-bp BamHI-Cohesive Single PETs
96
5.2.3.4
PET Concatenation
97
5.2.3.5
Purification of Concatenated PETs
97
5.2.3.6
Cloning Concatenated PETs in pZErO-1 Vector
98
5.2.3.7
Transform Cells
99
5.2.3.8
Carry out QC on GIS-PET Sequencing Library
99
X
Contents
5.2.4
ConstractìonofdiPETsfor454-Sequencing
200
5.2.4.1
Single-PET Plasmid
DNA
Preparation
100
5.2.4.2
Bse
RI
Linearization of Single-PET Plasmid
DNA 100
5.2.4.3
BamHI Digestion to Release Asymmetric PETs
101
5.2.4.4
Recovery and Quantitation of Purified Asymmetric PETs
101
5.2.4.5
Formation of diPETs
102
5.3
Data Analysis
103
5.4
Discussion
103
5.5
Perspectives
105
References
109
6
High-Throughput Functional Screening of Genes In
Planta
113
Thomas
Berberich,
Yoshihiro Takahashi, Hiromasa Saitoh, and
Ryohei Terauchi
6.1
Introduction
113
6.2
Methods and Protocols
115
6.2.1
Extraction of Total
RNA
116
6.2.2
Purification of Poly(A)+-mRNA
119
6.2.3
Synthesis of cDNA and Ligation to Binary, PVX-Based
Expression Vectors
121
6.2.4
Amplification of the cDNA Library in
E. coli
124
6.2.5
Transformation of cDNA library into Agrobacterium
tumefaciens Cells
126
6.2.5.1
Preparation of Competent Agrobacterium tumefaciens
Cells
326
6.2.6
Toothpick Inoculation of Leaves
128
6.2.7
Agroinfiltration
129
6.2.8
Recovery of the cDNA Fragments
130
6.3
Application of the Technology
131
6.4
Perspectives
133
References
134
7
Microarrays as Tools to Decipher
Transcriptomes
in Symbiotic
Interactions
137
Helge Küster
and
Anke
Becker
7.1
Introduction
137
7.2
Methods and Protocols
143
7.2.1
Spotting and Storage of
MtlĎkOLIl/MtlókOLIlPlus
70mer
Oligonucleotide Microarrays
143
7'.2.2
Synthesis of Targets by Indirect Reverse Transcription
Cy-Labeling
144
7.2.2.1
Components Stored at
-20 °С 144
7.2.2.2
Components Stored at
4
JC
(-20 °С
after Aliquoting
in
1/10
Volumes)
145
7.2.2.3
Components Stored at Room Temperature
145
Contents
XI
7.2.2.4 Reverse Transcription
of
Total
RNA
to obtain
Aminoalłyl-
Labeled
First-Strand cDNA
145
7.2.2.5
Hydrolysis of
RNA
246
7.2.2.6
Clean-Up of
Aminoalłyl-
Labeled First-Strand cDNA
146
7.2.2.7
Coupling of Fluorescent Dyes to Aminoallyl-Labeled
First-Strand cDNA
147
7.2.2.8
Quenching of all Remaining NHS Esters
147
7.2.2.9
Clean-up of Fluorescently Labeled Targets
147
7.2.2.10
Quality Control of Fluorescently Labeled Targets
148
7.2.3
Pre-Processing, Hybridization and Scanning of
MtlókOLIl/
MtlókOLIlPlus
Microarrays
148
7.2.4
Handling and Evaluation of Microarray Data
150
7.3
Applications of the Technology
151
7.3.1
Microarray-Based Identification of Medicago truncatula Genes
Induced during Different
Arbuscular Mycorrhizal
Interactions
151
7.3.2
Microarray-Based Identification of Medicago truncatula Genes
Activated during Nodulation and Mycorrhization
254
7.4
Perspectives
156
References
157
В
Gene-by-Cene Analysis
163
8
Genome-Wide Analysis of mRNA Expression by Fluorescent
Differential Display
165
Suping Zhou, Jonathan D. Meade, Samuel Nahashon, Blake R. Shester,
Jamie C.
Walden,
Then Cuo, Julia Z. Liang, Joshua G. Liang, and Peng Liang
8.1
Introduction
165
8.2
Methods and Protocols
169
8.2.1
Materials
169
8.2.1.1
Total
RNA
Isolation and Removal of Genomic
DNA
from
Total
RNA
169
8.2.1.2
Single-Strand cDNA Synthesis by Reverse Transcription
170
8.2.1.3
Fluorescent Differential Display-PCR (FDD-PCR)
270
8.2.1.4
Gel Electrophoresis
270
8.2.1.5
Reamplification
of Selected Differentially Expressed Bands
171
8.2.1.6
Cloning of Reamplified PCR Products
171
8.2.1.7
Verification of Cloned PCR Products
271
8.2.1.8
Confirmation of Differential Gene Expression by Northern Blot
172
8.2.2
Methods
172
8.2.2.1
Total
RNA
Isolation and Removal of Genomic
DNA 172
8.2.2.2
Gel Preparation
274
8.2.2.3
RNA
Loading Sample Preparation
274
8.2.2.4
Single-Strand cDNA Synthesis by Reverse Transcription
174
8.2.2.5
Fluorescent Differential Display-PCR
175
XII Contents
8.2.2.6
Gel Electrophoresis
176
8.2.2.7
Reamplification
of Selected Differentially Expressed
cDNA Bands
177
8.2.2.8
Cloning of Reamplified PCR Products
179
8.2.2.9
Verification of the Cloned Inserts
180
8.2.2.10
Confirmation of Differential Gene Expression by Northern Blot
181
8.3
Applications of the Technology
183
8.4
Perspectives
284
References
285
9
Real-Time Quantitation of MicroRNAs by TaqMan
MicroRNA Assays
287
Toni
L
Ceccardi,
Marianna M.
Goldrick, Peifeng Ren, Rick
С
Conrad, and
Caifu
Chen
9.1
Introduction
187
9.1.1
What are microRNAs?
287
9.1.2
Why are Researchers Interested?
288
9.1.3
Current Technologies for miRNA Quantitation
289
9.2
Methods and Protocols
290
9.2.1
Bioinformatic Tools for miRNA Discovery
290
9.2.2
MicroRNA Isolation from Plants
292
9.2.2.1
Extraction from Plant Tissue
192
9.2.2.2
PCR Directly from Cells in Culture
293
9.2.3
Description of TaqMan MicroRNA Assays
194
9.2.3.1
Principle of TaqMan MicroRNA Assays
194
9.2.3.2
Performing the TaqMan MicroRNA Assay
295
9.2.4
Data Normalization
296
9.3
Applications of the Technology
297
9.3.1
Quantitation of miRNAs
197
9.3.2
Absolute Quantitation of miRNAs
298
9.3.3
Expression Profiling of miRNAs
298
9.3.4
Verification of Predicted Novel miRNAs
299
9.3.5
MicroRNAs in Plant Growth and Development
299
9.3.6
Discovery of miRNA
Biomarkers
202
9.3.7
Discovery and Validation of Plant miRNA Targets
202
9.4
Perspectives
202
References
203
II Gene Silencing, Mutation Analysis and Functional Genomics
207
10
RNA
Interference
209
Chris A. Brosnan, Emily J. McCallum,
José
R.
Botella,
and Bernard
J.
Carroll
10.1
Introduction
209
10.2
Methods and Protocols
213
Contents XIII
10.3
Applications of the Technology
216
10.3.1
Targeting
Transgenes
in Arabidopsis using RNAi
216
10.3.2
Tissue-Specific
RNA
Silencing of an Endogenous Gene
in Tobacco
218
10.3.3
Advantages of Using RNAi in Plant Functional
Genomics
219
10.4
Perspectives
219
References
220
ΊΊ
Extending Functional Cenomics: VICS for Model and Crop Plants
227
Steven Bernacki, John Richard Tuttle, Nooduan Muangsan, and
Dominique Robertson
11.1
Introduction
227
11.2
Methods and Protocols
232
11.2.1
Constructing Geminiviras VIGS Vectors
232
11.2.1.1
Structure of Geminiviras Plasmids
233
11.2.1.2
Construction of an
ARI
Replacement Vector
234
11.2.1.3
Construction of an Insertion Vector
235
11.2.2
Silencing an Endogenous Gene
236
11.2.2.1
Visible Markers for Testing and Optimizing VIGS
236
11.2.2.2
Cloning a Target Gene Fragment(s) into the CaLCuVAiChll
Vector
237
11.2.2.3
Plant Preparation
237
11.2.2.4
Microprojectile Bombardment
238
11.2.2.5
Assessment of VIGS
239
11.3
Applications of the Technology
241
11.4
Perspectives
242
References
243
12
TILLING: A Reverse Genetics and a Functional Genomics Tool
in Soybean
251
Khalid
Mekšem,
Shiming Liu, Xiao Hong Liu, Azizjamai, Melissa Coellner
Mitchum, Abdelhafid Bendahmane, and Tarik El-Mellouki
12.1
Introduction
251
12.2
Methods and Protocols
252
12.2.1
Production of Suitable Mutant Population for TILLING
252
12.2.2 DNA
Extraction and
DNA
Construction of Pools
253
12.2.3
TILLING Screening for Mutations
254
12.2.3.1
Gene-Specific Primers for TILLING
254
12.2.3.2
PCR Amplification and Heteroduplex Formation
255
12.2.3.3
МІЗ
-Tailed
PCR Amplification for TILLING
255
12.2.3.4
Endonuclease Digestion and Purification of the Amplified
DNA 256
12.2.3.5
Gel Electrophoresis and Image Analysis
257
12.3
Applications of TILLING to Soybean
258
XIV Contents
12.3.1 Mutation Discovery,
Density
and Distribution in
two Mutagenized
Soybean Populations
258
12.3.2
Confirmation and Segregation Patterns of TILLING Mutations
in Soybean
260
12.4
Discussion and Perspectives
262
References
264
13
Transposon Tagging in Cereal Crops
267
Liza]. Conrad, Kazuhiro Kikuchi, and Thomas P. Brutnell
13.1
Insertional Mutagenesis in Plants
268
13.2
Transposon Tagging in Maize
270
13.2.1
Mutator Insertional Mutagenesis
270
13.2.2
Activator/Dissociation Mutagenesis
274
13.2.2.1
Forward Genetics
274
13.2.2.2
Reverse Genetics in Maize Using Ds
275
13.3
Large-Scale Reverse Genetics in Rice
275
13.3.1
Tosi
7
in Rice
276
13.3.2
The Maize
Ас
/Ds
Transposons
in Rice
277
13.3.3
En/Spm
279
13.4
Ас
/Ds
Transposon Tagging in Barley
280
13.5
Future Direction of Tagging in Cereals
281
13.5.1
Potential for an Endogenous Candystripel Tagging System
in Sorghum
281
13.5.2
Transposon-Mediated Deletions in Maize
282
13.5.3
Future Tagging Resources in Rice
282
13.5.4
Saturation Mutagenesis
282
References
282
14
Fast Neutron Mutagenesis for Functional Cenomics
291
Christian Rogers and
Сі
/es
Oldroyd
14.1
Introduction
291
14.1.1
Advantages of Fast Neutron Mutagenesis
291
14.1.2
Features of Fast Neutron Mutagenesis
292
14.1.3
Fast Neutron Mutagenesis for Reverse Genetics
294
14.2
Methods and Protocols
294
14.2.1
Screening Strategies
294
14.2.2
Automation
297
14.2.3
Establishing the Populations
298
14.2.4
Pooling Strategies
299
14.2.5
Characterization of the Populations
301
14.3
Applications of the Technology
301
14.3.1
Targeting Small Genes
302
14.3.2
Deletions Can Span Multiple Genes
303
14.3.3
Fast Neutron Reverse Genetics for Crop Improvement
303
Contents XV
14.4
Perspectives
304
References
304
III Computational Analysis
307
15
Bioinformatics Tools to Discover Co-Expressed
Cenes
in Plants
309
Yoshiyuki Ogata, Nozomu Sakurai, Nicholas J.
Provali,
Dirk Steinhauser,
and Leonard
Krall
15.1
Introduction
309
15.2
The Expression Angler Tool of the Botany Array Resource
311
15.2.1
Methods and Protocols
311
15.2.2
Applications of the Technology
317
15.2.3
Perspectives
319
15.3
The CSB.DB Tool
320
15.3.1
Introduction
320
15.3.2
Methods and Protocols
321
15.3.2.1
Simple Protocol for the Use of CSB.DB
322
15.4
The KaPPA-View
2:
Co-Expression Analysis on the Plant Metabolic
Pathway Maps
324
15.4.1
Introduction
324
15.4.2
Application of the Technology
324
15.4.3
Methods and Protocols
326
15.4.4
Perspectives
328
15.5
The KAGIANATool for Co-Expression Network Analysis
of Arabidopsis Genes
328
15.5.1
Introduction
328
15.5.2
Methods and Protocols
329
15.5.2.1
Initial Setting
329
15.5.2.2
Retrieval of Co-Expressed Genes
329
15.5.2.3
The Other Tools of KAGIANA
330
15.5.3
Perspective
331
References
331
16
AthaMap, a Database for the Identification and Analysis of Transcription
Factor Binding Sites in the Arabidopsis thaliana Genome
337
Reinhard Hehl
16.1
Introduction
337
16.2
Methods and Applications
339
16.2.1
Using the Web Interface at http://www.athamap.de/
339
16.2.1.1
The Search Function
339
16.2.1.2
Co-Localization Analysis
342
16.2.1.3
Gene Analysis
343
16.2.1.4
External links
344
References
345
XVI Contents
17
Structural
Phylogenomic Inference of Plant Gene Function
347
Nandini Krishnamurthy, Jim Leebens-Mack, and
Kimmen
Sjölander
17.1
Introduction
347
17.2
Challenges in Protein Function Prediction
349
17.2.1
Gene Duplication
350
17.2.2
Domain Shuffling
350
17.2.3
Speciation
352
17.2
A Propagation of Existing Annotation Errors
352
17.3
The Nomenclature of Homology
352
17.4
Structural Phylogenomic Inference of Function
354
17.5
Recommended Protocols for a Structural Phylogenomic Pipeline
356
17.5.1
Step
1: Homolog
Selection
357
17.5.2
Step
2:
Constructing and Analyzing a Multiple Sequence
Alignment
358
17.5.3
Step
3:
Constructing and Analyzing a Phylogenetic Tree
359
17.5.4
Step
4:
Predicting Function using a Phylogenetic Tree
361
17.6
Web Servers and Databases useful in Phylogenomic Inference
362
17.7
Discussion
363
References
364
18
Structural, Functional, and Comparative Annotation of Plant
Genomes
373
Françoise
Thibaud-Nissen, Jennifer Wortman,
С
Robin Buell, and Wei Zhu
18.1
Introduction
373
18.2
Methods, Protocols, and Applications
374
18.2.1
Structural Annotation
374
18.2.1.1
Cognate Transcript Sequences are the Most Reliable Data Available
378
18.2.1.2 Ab
initio Gene Finders are Good
ORF
Finders
379
18.2.1.3
Integrated Approaches are Ideal Solutions for the Automated
Gene Prediction
379
18.2.1.4
Gene Prediction is an Iterative Process
380
18.2.1.5
Manual duration is still an Indispensable Process
in Gene Prediction
380
18.2.1.6
Other Considerations for Gene Prediction
380
18.2.2
Functional Annotation
381
18.2.2.1
Sequence Similarity
381
18.2.2.2
Domain Searches
382
18.2.2.3
Phylogenomics
382
18.2.2.4
Expression Data
383
18.2.3
Comparative Annotation
383
18.2.3.1
Comparative Annotation Using Transcripts
383
18.2.3.2
Comparative Genomics Using Genome Sequences
385
18.2.3.3
Algorithms for Comparative Genomics
386
18.3
Perspectives
387
References
389
Contents
XVII
19
Large-Scale Cenomic Sequence Comparison and
Cene
Identification
with ClustDB
397
Jürgen
Kleffe
19.1
Introduction
397
19.2
Methods and Protocols
401
19.2.1
Reading Sequences
401
19.2.2
Substring Clusters
402
19.2.3
Maximally Extended Pairs of Common Substrings
405
19.2.4
Match Extension with Errors
406
19.2.5
Complete Matches
407
19.2.6
Reference Query Problems
407
19.2.7
Complementary Sequences
408
19.2.8
Handling Ambiguity Letter Codes
408
19.2.9
Sequence Clusters
409
19.2.10
Memory Analysis
410
19.3
Applications
410
19.3.1
Deriving Clusters of Identical Plant ESTs
411
19.3.2
Deriving Substring Clusters for All Plant ESTs
412
19.3.3
Checking the TIGR Medicago
ВАС
Assembly
412
19.4
Perspectives
413
References
415
IV Functional Cenomics and Emerging Technologies
417
20
Nanotechnologies and Fluorescent Proteins
fòr
in
pianta
Functional
Cenomics
419
C. Neal Stewart Jr.
20.1
Introduction
419
20.2
Green Fluorescent Protein
420
20.3
Protocol: Seeing GFP in Transgenic Plants
424
20.4
Nanotechnology for Monitoring Gene Expression
425
20.4.1
Aptamers and Quantum Dots
425
20.4.2
Molecular Beacons
425
20.4.3
Split GFP Tagging and Detection
426
20.5
Barriers to Implementation
427
20.6
Conclusions
427
References
428
21
New Frontiers in Plant Functional Cenomics Using Next
Generation Sequencing Technologies
431
Robert
С
Nutter
21.1
Introduction
433
21.1.1
Advent of Massively Parallel Sequencing Systems
431
21.1.2
Overview of the Sequencing by Synthesis System
432
XVIII
Contents
21.1.3
Overview of Single Base Extension System
431
21.1.4
Overview of the (SOliD) System
433
21.2
library Generation
434
21.3
Emulsion PCR
436
21.3.1
Bead Purification
436
21.3.2
Bead Deposition
437
21.4
Sequencing by Iigation
437
21.5
Base Calling
439
21.6
Potential Applications
440
21.6.1
Resequencing
440
21.6.2
De novo
Sequencing
440
21.6.3
Gene Expression via Sequence Tags
441
21.6.4
Other Tag-Based Applications
443
21.7
Conclusions
444
References
444
22 454
Sequencing: The Next Generation Tool for Functional
Cenomics
447
Lei Dia, Jan Frederik
Simons, Maithreyan Srinivasan, Thomas Jarvie,
Bruce Taillon, and Michael Egholm
22.1
Introduction
448
22.2
Methods and Protocols
450
22.2.1 DNA
Library Preparation
450
22.2.1.1
Option A: Nebulized library Procedure
451
22.2.1.2
Option B: Amplicon library Procedure
454
22.2.2
Emulsion PCR
456
22.2.3
Loading of PTP and Instrument Run
460
22.2.4
Data Analysis
467
22.2.4.1
Whole Genome Assembly
467
22.2.4.2
Resequencing and Mutation Detection
469
22.2.4.3
Ultra-deep Sequencing
469
22.3
Applications of the Technology
470
References
472
Glossary
477
Index
537 |
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| title | The handbook of plant functional genomics concepts and protocols |
| title_auth | The handbook of plant functional genomics concepts and protocols |
| title_exact_search | The handbook of plant functional genomics concepts and protocols |
| title_exact_search_txtP | The handbook of plant functional genomics concepts and protocols |
| title_full | The handbook of plant functional genomics concepts and protocols ed. by Günter Kahl ... |
| title_fullStr | The handbook of plant functional genomics concepts and protocols ed. by Günter Kahl ... |
| title_full_unstemmed | The handbook of plant functional genomics concepts and protocols ed. by Günter Kahl ... |
| title_short | The handbook of plant functional genomics |
| title_sort | the handbook of plant functional genomics concepts and protocols |
| title_sub | concepts and protocols |
| topic | Plant genetics blmsh Plant genomes blmsh Gentechnologie (DE-588)4071722-7 gnd Pflanzen (DE-588)4045539-7 gnd |
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