Ralph Rapley, Ph.D. Department of Biosciences, University of Hertfordshire, Hatfield, UKElectrical Engineers, Mechanical Engineers, Automation Engineers, Optical Industry, Students in Electrical Engineering, Students in Physics, Robotics Engineers, Automation Industry, Sensor Industry, Automobile Industry, Aircraft and Space Industry, Company Libraries, University Institutes and Libraries, Industry.

“It will be of immense value to students, biotechnologists, and scientists in the pharmaceutical industry. This update absolutely justifies replacement of the first edition.”  (Doody’s, 24 August 2012)

• Preface ix Contributors xi1 Overview of Genotyping 1Monica Bayes and Ivo Glynne GutIntroduction 1Methods for interrogating SNPs 3Commercial platforms for SNP genotyping 7Practical recommendations 11SNP databases 14Methylation analysis 15Copy number variation analysis 16Second generation sequencing technologies 17Conclusions 18References 192 DNA Chip Analysis in Genome Discovery 24Ross N. Nazar and Jane RobbIntroduction 24Interrogating a genome 25Cross-species hybridization 28Comparative genomic hybridization and microarray-based genotyping 31Barcodes, DNA microarrays and organism identification 34Concluding remarks 36References 373 qPCR, Theory, Reliability and Use in Molecular Analysis 43Jamie Murphy and Stephen A. BustinSample preparation 44RNA quality 45Reagents 46Assay design 47Transparency of published data 51Further considerations 52Conclusion 52References 524 DNA Analysis in Droplet-Based Microfluidic Devices 56Pinar Ozdemir and Yonghao ZhangIntroduction 56Continuous-flow microPCR chips 58PCR inhibition and carryover contamination 62PCR in droplets 65Conclusions 73References 755 High-Resolution Melt Profiling 81Steven F. Dobrowolski and Carl T. WittwerIntroduction 81Basic concepts of melt profiling 82HRMP and polymerase chain reaction 84DNA specimens and HRMP 89Determining a temperature window for melting 90Dyes and platforms for melt profiling 92Scanning PCR products for sequence variation 93Genotyping with high-resolution melt profiling 102Other applications of HRMP 108Final notes 109References 1106 Massively Parallel Sequencing 114Tracy Tucker, Marco Marra and Jan M. FriedmanSanger sequencing 114Massively parallel sequencing 114Commercially available massively parallel sequencers 115Future technologies 122Paired-end or mate-paired reads 123Target-enrichment strategies for MPS 124Applications of MPS 125Summary 131References 1327 Aptamers for Analysis: Nucleic Acids Ligands in the Post-Genomic Era 135Pedro Nadal, Alessandro Pinto, Marketa Svobodova and Ciara K. O'SullivanIntroduction 135SELEX 136Aptamers in analysis 147Imaging with aptamers 157Conclusions, outlooks and perspectives 160References 1618 Use of Nanotechnology for Enhancing of Cancer Biomarker Discovery and Analysis: A Molecular Approach 175Farid E. AhmedIntroduction 175Proteomics and nanotechnology 176Nanoscale multicomponent separation 176Nanoscale protein detection strategies 178Surface-enhanced Raman scattering (SERS) 182References 1889 Chip-Based Proteomics 193Julian Bailes, Andrew Milnthorpe, Sandra Smieszek and Mikhail SolovievIntroduction 193Lab-on-a-chip 194Arrays 196Chip-based mass spectrometry 203Surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) chip instruments 205Microfluidics 207Conclusion 209References 21110 Antibody Microarrays in Proteome Profiling 219Mohamed Saiel Saeed Alhamdani and Jorg D. HoheiselIntroduction 219Technical aspects 220Antibody array applications 226Summary 229Acknowledgements 229References 22911 Biomarker Detection and Molecular Profiling by Multiplex Microbead Suspension Array Based Immunoproteomics 244V. V. Krishhan, Imran H. Khan and Paul A. LuciwIntroduction 244Principles of microbead-based multiplexing 245Experimental aspects of the multiplex microbead assay 247Multiplex microbead assay design and comparison with other methods 250Applications of the multiplex microbead assay system for biomedical research and clinical studies 252Selected investigational fields for multiplex analysis and examples of applications 255Challenges and current limitations 255Summary and future directions 259Acknowledgements 261References 26112 Mass Spectrometry in Metabolomics 271William J. Griffiths and Yuqin WangIntroduction 271Sample collection and preparation 272Data acquisition 274Data analysis 279Applications 283Conclusion 294Acknowledgement 294References 295Index 299

Ralph Rapley, Ph.D. Department of Biosciences, University of Hertfordshire, Hatfield, UKElectrical Engineers, Mechanical Engineers, Automation Engineers, Optical Industry, Students in Electrical Engineering, Students in Physics, Robotics Engineers, Automation Industry, Sensor Industry, Automobile Industry, Aircraft and Space Industry, Company Libraries, University Institutes and Libraries, Industry.

“It will be of immense value to students, biotechnologists, and scientists in the pharmaceutical industry. This update absolutely justifies replacement of the first edition.”  (Doody’s, 24 August 2012)

• Preface ix Contributors xi1 Overview of Genotyping 1Monica Bayes and Ivo Glynne GutIntroduction 1Methods for interrogating SNPs 3Commercial platforms for SNP genotyping 7Practical recommendations 11SNP databases 14Methylation analysis 15Copy number variation analysis 16Second generation sequencing technologies 17Conclusions 18References 192 DNA Chip Analysis in Genome Discovery 24Ross N. Nazar and Jane RobbIntroduction 24Interrogating a genome 25Cross-species hybridization 28Comparative genomic hybridization and microarray-based genotyping 31Barcodes, DNA microarrays and organism identification 34Concluding remarks 36References 373 qPCR, Theory, Reliability and Use in Molecular Analysis 43Jamie Murphy and Stephen A. BustinSample preparation 44RNA quality 45Reagents 46Assay design 47Transparency of published data 51Further considerations 52Conclusion 52References 524 DNA Analysis in Droplet-Based Microfluidic Devices 56Pinar Ozdemir and Yonghao ZhangIntroduction 56Continuous-flow microPCR chips 58PCR inhibition and carryover contamination 62PCR in droplets 65Conclusions 73References 755 High-Resolution Melt Profiling 81Steven F. Dobrowolski and Carl T. WittwerIntroduction 81Basic concepts of melt profiling 82HRMP and polymerase chain reaction 84DNA specimens and HRMP 89Determining a temperature window for melting 90Dyes and platforms for melt profiling 92Scanning PCR products for sequence variation 93Genotyping with high-resolution melt profiling 102Other applications of HRMP 108Final notes 109References 1106 Massively Parallel Sequencing 114Tracy Tucker, Marco Marra and Jan M. FriedmanSanger sequencing 114Massively parallel sequencing 114Commercially available massively parallel sequencers 115Future technologies 122Paired-end or mate-paired reads 123Target-enrichment strategies for MPS 124Applications of MPS 125Summary 131References 1327 Aptamers for Analysis: Nucleic Acids Ligands in the Post-Genomic Era 135Pedro Nadal, Alessandro Pinto, Marketa Svobodova and Ciara K. O'SullivanIntroduction 135SELEX 136Aptamers in analysis 147Imaging with aptamers 157Conclusions, outlooks and perspectives 160References 1618 Use of Nanotechnology for Enhancing of Cancer Biomarker Discovery and Analysis: A Molecular Approach 175Farid E. AhmedIntroduction 175Proteomics and nanotechnology 176Nanoscale multicomponent separation 176Nanoscale protein detection strategies 178Surface-enhanced Raman scattering (SERS) 182References 1889 Chip-Based Proteomics 193Julian Bailes, Andrew Milnthorpe, Sandra Smieszek and Mikhail SolovievIntroduction 193Lab-on-a-chip 194Arrays 196Chip-based mass spectrometry 203Surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) chip instruments 205Microfluidics 207Conclusion 209References 21110 Antibody Microarrays in Proteome Profiling 219Mohamed Saiel Saeed Alhamdani and Jorg D. HoheiselIntroduction 219Technical aspects 220Antibody array applications 226Summary 229Acknowledgements 229References 22911 Biomarker Detection and Molecular Profiling by Multiplex Microbead Suspension Array Based Immunoproteomics 244V. V. Krishhan, Imran H. Khan and Paul A. LuciwIntroduction 244Principles of microbead-based multiplexing 245Experimental aspects of the multiplex microbead assay 247Multiplex microbead assay design and comparison with other methods 250Applications of the multiplex microbead assay system for biomedical research and clinical studies 252Selected investigational fields for multiplex analysis and examples of applications 255Challenges and current limitations 255Summary and future directions 259Acknowledgements 261References 26112 Mass Spectrometry in Metabolomics 271William J. Griffiths and Yuqin WangIntroduction 271Sample collection and preparation 272Data acquisition 274Data analysis 279Applications 283Conclusion 294Acknowledgement 294References 295Index 299

Ralph Rapley, Ph.D. Department of Biosciences, University of Hertfordshire, Hatfield, UKElectrical Engineers, Mechanical Engineers, Automation Engineers, Optical Industry, Students in Electrical Engineering, Students in Physics, Robotics Engineers, Automation Industry, Sensor Industry, Automobile Industry, Aircraft and Space Industry, Company Libraries, University Institutes and Libraries, Industry.

“It will be of immense value to students, biotechnologists, and scientists in the pharmaceutical industry. This update absolutely justifies replacement of the first edition.”  (Doody’s, 24 August 2012)

• Preface ix Contributors xi1 Overview of Genotyping 1Monica Bayes and Ivo Glynne GutIntroduction 1Methods for interrogating SNPs 3Commercial platforms for SNP genotyping 7Practical recommendations 11SNP databases 14Methylation analysis 15Copy number variation analysis 16Second generation sequencing technologies 17Conclusions 18References 192 DNA Chip Analysis in Genome Discovery 24Ross N. Nazar and Jane RobbIntroduction 24Interrogating a genome 25Cross-species hybridization 28Comparative genomic hybridization and microarray-based genotyping 31Barcodes, DNA microarrays and organism identification 34Concluding remarks 36References 373 qPCR, Theory, Reliability and Use in Molecular Analysis 43Jamie Murphy and Stephen A. BustinSample preparation 44RNA quality 45Reagents 46Assay design 47Transparency of published data 51Further considerations 52Conclusion 52References 524 DNA Analysis in Droplet-Based Microfluidic Devices 56Pinar Ozdemir and Yonghao ZhangIntroduction 56Continuous-flow microPCR chips 58PCR inhibition and carryover contamination 62PCR in droplets 65Conclusions 73References 755 High-Resolution Melt Profiling 81Steven F. Dobrowolski and Carl T. WittwerIntroduction 81Basic concepts of melt profiling 82HRMP and polymerase chain reaction 84DNA specimens and HRMP 89Determining a temperature window for melting 90Dyes and platforms for melt profiling 92Scanning PCR products for sequence variation 93Genotyping with high-resolution melt profiling 102Other applications of HRMP 108Final notes 109References 1106 Massively Parallel Sequencing 114Tracy Tucker, Marco Marra and Jan M. FriedmanSanger sequencing 114Massively parallel sequencing 114Commercially available massively parallel sequencers 115Future technologies 122Paired-end or mate-paired reads 123Target-enrichment strategies for MPS 124Applications of MPS 125Summary 131References 1327 Aptamers for Analysis: Nucleic Acids Ligands in the Post-Genomic Era 135Pedro Nadal, Alessandro Pinto, Marketa Svobodova and Ciara K. O'SullivanIntroduction 135SELEX 136Aptamers in analysis 147Imaging with aptamers 157Conclusions, outlooks and perspectives 160References 1618 Use of Nanotechnology for Enhancing of Cancer Biomarker Discovery and Analysis: A Molecular Approach 175Farid E. AhmedIntroduction 175Proteomics and nanotechnology 176Nanoscale multicomponent separation 176Nanoscale protein detection strategies 178Surface-enhanced Raman scattering (SERS) 182References 1889 Chip-Based Proteomics 193Julian Bailes, Andrew Milnthorpe, Sandra Smieszek and Mikhail SolovievIntroduction 193Lab-on-a-chip 194Arrays 196Chip-based mass spectrometry 203Surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) chip instruments 205Microfluidics 207Conclusion 209References 21110 Antibody Microarrays in Proteome Profiling 219Mohamed Saiel Saeed Alhamdani and Jorg D. HoheiselIntroduction 219Technical aspects 220Antibody array applications 226Summary 229Acknowledgements 229References 22911 Biomarker Detection and Molecular Profiling by Multiplex Microbead Suspension Array Based Immunoproteomics 244V. V. Krishhan, Imran H. Khan and Paul A. LuciwIntroduction 244Principles of microbead-based multiplexing 245Experimental aspects of the multiplex microbead assay 247Multiplex microbead assay design and comparison with other methods 250Applications of the multiplex microbead assay system for biomedical research and clinical studies 252Selected investigational fields for multiplex analysis and examples of applications 255Challenges and current limitations 255Summary and future directions 259Acknowledgements 261References 26112 Mass Spectrometry in Metabolomics 271William J. Griffiths and Yuqin WangIntroduction 271Sample collection and preparation 272Data acquisition 274Data analysis 279Applications 283Conclusion 294Acknowledgement 294References 295Index 299