Nucleic Acids Hybridization Modern Applications, 1st Edition

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vi Contents

2. Selective Suppression of Polymerase Chain Reaction

and Its Most Popular Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Sergey A. Lukyanov, Konstantin A. Lukyanov, Nadezhda G. Gurskaya,
Ekaterina A. Bogdanova, Anton A. Buzdin
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2. Selective PCR Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3. Preparation of ITR-Containing DNA Samples . . . . . . . . . . . . . . . 32
4. Strategy of Employment of SSP . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5. SSP-based Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5.1 Construction of cDNA Libraries from a Small Amount
of Biological Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
5.2 Detection of Differentially Expressed Genes . . . . . . . . . . . . . . 36
5.3 Search for 5′- and 3′-Terminal Fragments of cDNA . . . . . . . . 40
5.4 Search for Promoter Sites (Chromosome Walking) . . . . . . . . . 40
5.5 In Vitro Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
5.6 Multiplex PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
6. Detailed Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
6.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
6.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

3. Suppression Subtractive Hybridization . . . . . . . . . . . . . . . . . . . . . . . . 53

Sergey A. Lukyanov, Denis Rebrikov, Anton A. Buzdin
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
2. The Principle of Suppression Subtractive Hybridization . . . . . . . . 55
3. The Principle of Mirror-Oriented Selection . . . . . . . . . . . . . . . . . . 57
4. Technical Comments and Considerations . . . . . . . . . . . . . . . . . . . . 59
4.1 Normalization Step and the Efficiency of SSH . . . . . . . . . . . . 59
4.2 Differential Screening of the Subtracted Libraries . . . . . . . . . . 61
4.3 Starting RNA Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
4.4 Size of cDNA Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
5. Detailed Protocols: Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.1 Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.2 Buffers and Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
6. Detailed Protocols: Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
6.1 Preparation of Subtracted cDNA or Genomic
DNA Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
6.2 Mirror Orientation Selection . . . . . . . . . . . . . . . . . . . . . . . . . . 70
6.3 Cloning of Subtracted cDNAs . . . . . . . . . . . . . . . . . . . . . . . . . 74
6.4 Differential Screening of the Subtracted cDNA Library . . . . . 74
7. Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Contents vii

4. Stem-Loop Oligonucleotides as Hybridization Probes

and Their Practical Use in Molecular Biology and Biomedicine . . . . . 85
Anton A. Buzdin, Sergey A. Lukyanov
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
2. Molecular Beacons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
3. Stem-Loop DNA Probes on Microarrays . . . . . . . . . . . . . . . . . . . . 92
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

5. Normalization of cDNA Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Alex S. Shcheglov, Pavel A. Zhulidov, Ekaterina A. Bogdanova,
Dmitry A. Shagin
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
1.1 Normalized cDNA Libraries: What are they needed for? . . . . . 98
1.2 Evaluation of cDNA Library Normalization Efficacy . . . . . . . 100
1.3 Basic Approaches to Generate Normalized
cDNA Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
2. Methods of cDNA Normalization . . . . . . . . . . . . . . . . . . . . . . . . . 102
2.1 cDNA Normalization by Means of Hydroxyapatite
Column Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
2.2 Generation of Normalized Full-Length-Enriched cDNA
Libraries Using DNA Immobilization on a Solid Support . . . . 104
2.3 Normalization of Full-Length cDNA with the
use of Biotinylated RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
2.4 Normalization of Fragmented cDNA by Means
of Selective Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
2.5 cDNA Normalization Using Frequent-Cutter
Restriction Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
2.6 Normalization of Full-Length-Enriched cDNA
with Duplex-Specific Nuclease (DSN Normalization) . . . . . . . 109
3. cDNA Preparation for DSN Normalization . . . . . . . . . . . . . . . . . . 111
3.1 RNA Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
3.2 cDNA Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
3.3 cDNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
4. DSN Normalization Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
4.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
4.2 cDNA Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
4.3 Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
4.4 DSN Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
4.5 First Amplification of Normalized cDNA . . . . . . . . . . . . . . . . 117
4.6 Preliminary Analysis of the Normalization Results . . . . . . . . . 119
4.7 Second Amplification of Normalized cDNA . . . . . . . . . . . . . . 121
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
viii Contents

6. Primer Extension Enrichment Reaction (PEER)

and Other Methods for Difference Screening . . . . . . . . . . . . . . . . . . . 125
Lilia M. Ganova-Raeva
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
2. Primer Extension Enrichment Reaction . . . . . . . . . . . . . . . . . . . . . 128
2.1 Method Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
2.2 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
2.3 PEER Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
3. Other Subtraction and Hybridization Based Methods
for Difference Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
3.1 Differential Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
3.2 Subtractive Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
3.3 Subtractive Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
3.4 Differential Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
3.5 AFLP, SAGE/CAGE, GSTs, and DARFA . . . . . . . . . . . . . . . 142
3.6 Representational Differences Analysis . . . . . . . . . . . . . . . . . . 143
3.7 SPAD–RDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
3.8 Enzymatic Degradation Subtractions (EDS, LCS,
DSC, NSC, UDG/USA, and CODE) . . . . . . . . . . . . . . . . . . . 146
3.9 Suppression Subtraction Hybridization . . . . . . . . . . . . . . . . . 150
3.10 Selective Amplification Via Biotin and
Restriction-Mediated Enrichment . . . . . . . . . . . . . . . . . . . . . 153
3.11 DNA Enrichment by Allele-Specific
Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
3.12 Methods Combining the use of SSH
and Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
3.13 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

7. Subtractive Hybridization with Covalently

Modified Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Shi-Lung Lin, Donald Chang, Joseph D. Miller, Shao-Yao Ying
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
2. Subtractive Hybridization Methods . . . . . . . . . . . . . . . . . . . . . . . . 169
3. Covalent Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
4. Subtractive Hybridization with Covalently Modified
Subtracters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
5. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
6. Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
6.1 Preparation of Subtracter and Tester DNA Libraries . . . . . . 180
6.2 Covalent Modification of Subtracter DNAs . . . . . . . . . . . . . 180
6.3 Subtractive Hybridization and CHS–PCR Amplification . . . 181
Contents ix

6.4 Covalent Binding Efficiency and Subtractive

Stringency of CHS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
6.5 Identification of Genomic Deletion Using CHS . . . . . . . . . . . 184
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

8. Coincidence Cloning: Robust Technique for Isolation

of Common Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Anton A. Buzdin
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
2. Cloning Selection of Heteroduplexes . . . . . . . . . . . . . . . . . . . . . . . 191
3. Physical Separation of Hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
4. PCR-only-based Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
5. Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
6. Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
6.1 Cloning Similarities in Genomic DNAs . . . . . . . . . . . . . . . . . . 202
6.2 Cloning and Presice Mapping of Transcribed
Repetitive Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
6.3 Finding Methylated or Unmethylated CpGs
in Large Genomic Contigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

9. DNA Hybridization in Solution for Mutation Detection . . . . . . . . . . 211

Anton A. Buzdin
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
2. Chemical Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
3. Enzymatic Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
3.1 Nuclease-Based Mutation Scanning . . . . . . . . . . . . . . . . . . . . . 218
3.2 Allele-Specific PCR-Based Approaches . . . . . . . . . . . . . . . . . . 229
3.3 Other Enzymatic Approaches for Mutation
Scanning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
4. Physical Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
5. Bioinformatical Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

10. Current Attempts to Improve the Specificity of Nucleic Acids

Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Anton A. Buzdin
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
2. Improving Hybridization Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . 244
2.1 Simplification of Hybridizing Mixtures . . . . . . . . . . . . . . . . . . 246
2.2 Chemical Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
3. Improving Selection of Perfectly Matched Hybrids . . . . . . . . . . . . 249
x Contents

4. Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
4.1 Targeted Genomic Difference Analysis . . . . . . . . . . . . . . . . . . 255
4.2 Using Competitor DNA to Decrease
the Background of Genomic Repeats . . . . . . . . . . . . . . . . . . . 258
4.3 Mispaired DNA Rejection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

11. Concepts on Microarray Design for Genome

and Transcriptome Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Helder I. Nakaya, Eduardo M. Reis, Sergio Verjovski-Almeida
1. Building a Microarray Chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
1.1 Spotted DNA Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
1.2 In situ Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
2. Selecting the Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
2.1 Gene-Oriented Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
2.2 Epigenomic Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
2.3 Tiling Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
3. Specific Question, Specific Chip . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
3.1 Transcriptional Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
3.2 Comparative Genome Hybridization . . . . . . . . . . . . . . . . . . . . 282
3.3 Alternative Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
3.4 Transcriptome Annotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
3.5 Small MicroRNA Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
3.6 Methylation Pattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
3.7 ChIP-Chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
3.8 Genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
3.9 Intronic Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
CONTRIBUTORS

Ekaterina A. Bogdanova
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Moscow 117997, Miklukho-Maklaya 16/10, Russia

Anton A. Buzdin
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Moscow 117997, Miklukho-Maklaya 16/10, Russia

Nadezhda G. Gurskaya
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Moscow 117997, Miklukho-Maklaya 16/10, Russia

Konstantin A. Lukyanov
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Moscow 117997, Miklukho-Maklaya 16/10, Russia

Sergey A. Lukyanov
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Moscow 117997, Miklukho-Maklaya 16/10, Russia

Denis Rebrikov
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Moscow 117997, Miklukho-Maklaya 16/10, Russia

xi
xii Contributors

Dmitry A. Shagin
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Moscow 117997, Miklukho-Maklaya 16/10, Russia

Alex S. Shcheglov
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Moscow 117997, Miklukho-Maklaya 16/10, Russia

Pavel A. Zhulidov
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Moscow 117997, Miklukho-Maklaya 16/10, Russia

Donald Chang
Department of Cell and Neurobiology, Keck School of Medicine,
University of Southern California,
1333 San Pablo Street, BMT-403,
Los Angeles, CA 90033, USA

Shi-Lung Lin
Department of Cell and Neurobiology, Keck School of Medicine,
University of Southern California,
1333 San Pablo Street, BMT-403,
Los Angeles, CA 90033, USA

Joseph Miller
Department of Cell and Neurobiology, Keck School of Medicine,
University of Southern California,
1333 San Pablo Street, BMT-403,
Los Angeles, CA 90033, USA

Shao-Yao Ying
Department of Cell and Neurobiology, Keck School of Medicine,
University of Southern California,
1333 San Pablo Street, BMT-403,
Los Angeles, CA 90033, USA

Lilia M. Ganova-Raeva
Centers for Disease Control and Prevention
Division of Viral Hepatitis
1600 Clifton Rd. NE, MS A-33
Atlanta, Georgia 30329, USA
Contributors xiii

Helder I. Nakaya
Departamento de Bioquimica, Instituto de Quimica,
Universidade de São Paulo,
05508-900 São Paulo, SP, Brazil

Eduardo M. Reis
Departamento de Bioquimica, Instituto de Quimica,
Universidade de São Paulo,
05508-900 São Paulo, SP, Brazil

Sergio Verjovski-Almeida
Departamento de Bioquimica, Instituto de Quimica,
Universidade de São Paulo,
05508-900 São Paulo, SP, Brazil
ACKNOWLEDGMENTS

Anton A. Buzdin:
Many thanks to Professor Eugene D. Sverdlov for his fruitful discussion, inno-
vative ideas, and overall support of this project. Thanks to my colleagues,
friends, and family members for their help, patience, and understanding.
A. Buzdin was funded by the Molecular and Cellular Biology Program of the
Presidium of the Russian Academy of Sciences, by the personal grant from the
President of the Russian Federation, and by Russian Foundation for Basic
Research grants Nos. 05-04-48682-a, 2006.20034.
Sergey A. Lukyanov, Alex Shcheglov, Pavel Zhulidov, Ekaterina Bogdanova, Dmitry
Shagin:
Work on this book was supported by the Molecular and Cellular Biology
Program of the Russian Academy of Sciences and Evorogen JSC (Moscow,
Russia).
Lilia M. Ganova-Raeva:
Special thanks to Dr. Y. Khudyakov who contributed most to the PEER
backbone idea and has been relentlessly resourceful, helpful, and patient
throughout the development of the method. Thanks to Dr. H. Fields in whose
lab the PEER testing was initiated. Thanks to Dr. X. Zhang for great help with
the library screenings and to Dr. F. Cao for introducing better enzymes in the
protocol.
Shi-Lung Lin, Donald Chang, Joseph D. Miller, Shao-Yao Ying:
This study was supported by NIH/NCI grant CA-85722. Rhw CHS technology
is the property of University of Southern California and protected by US patent
numbers, 5,928,872 and 6,130,040.
xv
xvi Acknowledgments

Helder I. Nakaya, Eduardo M. Reis, Sergio Verjovski-Almeida:

The work in the authors’ laboratory was supported by grants and fellowships
from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and
Conselho Nacional do Desenvolvimento Científico e Tecnológico (CNPq),
Brazil.
PREFACE

Watson–Crick hybridization of complementary sequences in nucleic acids is one

of the most important fundamental processes necessary for molecular recogni-
tion in vivo, as well as for nucleic acid identification and isolation in vitro. This
book is devoted to a large family of in vitro DNA hybridization-based experi-
mental techniques. A wide spectrum of experimental tasks covered by these
approaches includes finding differential sequences in both genomic DNAs and
mRNAs, genome walking, multiplex PCR, cDNA library construction starting
from minute amount of total RNA, rapid amplification of cDNA 5′- and 3′-
ends, effective smoothing of the concentrations of rare and abundant transcripts
in cDNA libraries, recovery of promoter active repeats and differentially methy-
lated genomic DNA, identification of common sequences in genomic or cDNA
sources, new gene mapping, finding evolutionary conserved DNA and both sin-
gle-nucleotide and extended mutation discovery, or large-scale monitoring.
Several approaches, such as microarray hybridization, have become extremely
popular tools for specialists in biochemistry and biomedicine, whereas the poten-
tial of many other advantageous techniques seems to be underestimated now.
Analysis of differential gene expression requires application of global
approaches that represent a leading tool in postgenomic studies and include
transcriptome and proteome analysis, as well as methods allowing population-
wide sequence and functional polymorphism analysis. Central to these new
technologies are DNA chips designed for quantitative and qualitative uses.
Although they are very useful and widely distributed, many popular DNA
microarray techniques share a number of shortcomings:
1. The analysis is limited by a number of cDNAs/synthetic oligonucleotides
applied on the chip. This number is usually significantly lower than the total
xvii
xviii Preface

gene quantity of the organisms under study. It creates, therefore, a problem,

that many genes escape such an analysis.
2. General transcriptome-wide chip techniques in their actual state hardly
distinguish between different gene splice forms.
3. The expression of genes transcribed at low levels cannot be detected by using
standard microarray approaches.
4. cDNA-based chips do not differentiate between many gene family members
and/or between many transcripts containing repetitive DNA.
5. Microarray chips lack many natural RNAi, cDNAs, or synthetic oligonucleotides
and, therefore, cannot be used for the comprehensive study of gene expression
regulation at the level of RNA interference by small interfering RNAs.
However, most of these concerns can be effectively addressed by using specific
variants of microchip technology, thus making microarrays a truly universal
technique (see Chapter 11). Probably, the most important disadvantage of
closed systems such as microarrays is that they require preliminary genomic
sequence information in order to identify differentially expressed transcripts.
Open systems have the flexibility of identifying uncatalogued sequences.
Related experimental techniques, based on DNA hybridization in solution, may
be advantageous for many applications, starting from representative cDNA
library construction for expressed sequence tag (EST) sequencing, to the identi-
fication of evolutionary conserved sequences, differentially expressed genes, or
genomic deletions. Unique characteristics of many such techniques make them
powerful competitors for well-known approaches that are appreciated world-
wide like microarray and competitive genomic hybridizations. Nucleic acid
hybridization in solution has few general advantages over hybridization with
solid carrier-immobilized nucleic acids: faster hybridization kinetics, better
discrimination of proper hybrids, and their availability for further PCR amplifi-
cation and cloning. Among such in-solution hybridization methods, subtractive
hybridization is undoubtedly the most popular technique.
Many techniques have a low efficiency of identifying rare genes that are differ-
entially expressed. This problem is exacerbated when the change in expression level
of rare transcripts is small. Since genes expressed at low levels also play a role in
establishing differentiated phenotypes, their identification is essential for a com-
plete mechanistic understanding of cellular changes. The major advantage of sub-
tractive hybridization lies in the ability to identify differentially transcribed genes,
irrespective of the level of expression, in the absence of sequence information. In
addition to preparation of differential cDNA libraries, subtractive hybridization is
also extremely useful for identification of genomic DNA differences.
Coincidence cloning, on the contrary, makes it possible to identify sequences
which are common for all samples under comparison; cDNA normalization,
which is used for smoothing of rare and frequent transcript content in cDNA
libraries, may be extremely useful for representative EST library construction.
Moreover, several techniques deal with the large-scale DNA polymorphism
recovery, including identification of single nucleotide polymorphisms.
Preface xix

The international team of the authors of this book has tried both to elucidate
the current state of the art in hybridization techniques and to help the readers
in choosing an appropriate method for performing an experiment in the most
efficient way. Enclosed experimental protocols along with both comprehensive
and detailed method descriptions make this truly universal book useful to all
those interested in the modern life science methodologies.
CHAPTER 1

NUCLEIC ACIDS HYBRIDIZATION: POTENTIALS

AND LIMITATIONS

ANTON A. BUZDIN

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10

Miklukho-Maklaya, 117997 Moscow, Russia
Phone: +(7495) 3306329; Fax: +(7495) 3306538; E-mail: [email protected]

Abstract: Several nucleic acids hybridization-based approaches, such as microarray, competi-

tive genomic, and Southern or Northern blot hybridization, have become popular
tools for specialists in biochemistry and in biomedicine, and are now in routine use.
However, the potential of in-solution nucleic acids hybridization-based experimen-
tal techniques seems to be underestimated now. Examples are subtractive hybridiza-
tion (SH), which allows one to efficiently find differences in genomic DNAs or in
cDNA samples; coincidence cloning (CC), which, on the contrary, makes it possible
to identify sequences that are present in all the samples under comparison; cDNA
normalization, which is used for the smoothing of rare and frequent transcript con-
tent in cDNA libraries; and TILLING approach, which has demonstrated its great
potential for the reverse genetics studies. Finally, several techniques are aimed at the
large-scale recovery of DNA polymorphisms, including single nucleotide polymor-
phisms (SNPs). This book will focus on the above-mentioned and other recent
developments in the area of nucleic acids hybridization, including attempts to
improve its specificity. In this introductory chapter, I have tried to briefly character-
ize the current state of the art in in-solution nucleic acids hybridization techniques,
and to define their major principles and applications. The advantages and short-
comings of these techniques will be discussed here.

Keywords: Nucleic acids hybridization, cDNA library construction, EST, differentially expressed
genes, differential transcripts, differential sequence, microarray, competitive genomic
hybridization, subtractive hybridization, coincidence cloning, rare transcript, frequent
transcript, normalization, cDNA normalization, cDNA subtraction, genomic
subtraction, polymorphism recovery, mutation, single nucleotide polymorphism,
SNP, hybridization kinetics, hybridization rate, tracer, tester, driver, genome size,
genome complexity, representational differences analysis (RDA), subcloning, restric-
tion fragment length polymorphisms recovery, suppression subtractive hybridization,
SSH, genomic polymorphism, Sanger sequencing, mispaired nucleotides, mutant
strand, wild-type strand, mutant allele, wild-type allele, rapid amplification of cDNA
ends, RACE, differentially methylated.
1
A. Buzdin and S. Lukyanov (eds.), Nucleic Acids Hybridization, 1–28.
© 2007 Springer.
2 A. A. Buzdin
Abbreviations: BAC, bacterial artificial chromosome; CC, coincidence cloning; cDNA, comple-
mentary DNA; CHS, covalently hybridized subtraction; dNTP, deoxyribonu-
cleotidetriphosphate; EST, expressed sequence tag; GREM, genomic repeat
expression monitor; mRNA, messenger RNA; MOS, mirror orientation selection;
NGSCC, nonmethylated genomic sites coincidence cloning; PEER, primer exten-
sion enrichment reaction; PCR, polymerase chain reaction; PERT, phenol emul-
sion reassociation technique; RACE, rapid amplification of cDNA ends; RDA,
representative differences analysis; RFLP, restriction fragment length polymor-
phism; RNAi, interfering RNA; RT, reverse transcription; SAGE, serial analysis
of gene expression; SH, subtractive hybridization; SNP, single nucleotide poly-
morphism; SSH, suppression subtractive hybridization; YAC, yeast artificial
chromosome.

TABLE OF CONTENTS

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Cloning the Differences: Subtractive Hybridization . . . . . . . . . . . . . . . . . . 4
2.1 Birth of a Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2 PCR-assisted Subtractive Hybridization . . . . . . . . . . . . . . . . . . . . . . . 8
2.3 First Worldwide Success: Representational Differences Analysis . . . . 10
2.4 Further Improvements: Suppression Subtractive
Hybridization, Polymerase Chain Reaction Suppression
Effect, and Normalization of cDNA Libraries . . . . . . . . . . . . . . . . . 13
2.5 Covalently Hybridized Subtraction, Primer Extension
Enrichment Reaction, and other Promising Approaches
in Subtractive Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3. Finding Common DNA: Coincidence Cloning . . . . . . . . . . . . . . . . . . . . 20
4. Hybridization in Solution for the Recovery of
Genomic Polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

1. INTRODUCTION

Watson–Crick hybridization of complementary sequences in nucleic acids is one

of the most important fundamental processes necessary for molecular recogni-
tion in vivo (Watson and Crick 1953), as well as nucleic acid identification and
isolation in vitro (Southern 1992). The use of experimental techniques based on
DNA hybridization in solution is advantageous for many applications, starting
from representative complementary DNA (cDNA) library construction for
expressed sequence tag (EST) sequencing to the identification of evolutionary
conserved sequences, differentially expressed genes, or genomic deletions.
Unique characteristics of many such techniques make them powerful competitors