Current Protocols in Human Genetics

Current Protocols in Human Genetics

Online ISBN: 9780471142904

DOI: 10.1002/0471142905

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  1. Preface
  2. Foreword
  3. Selected Suppliers of Reagents and Equipment
  4. Chapter 1 Genetic Mapping
    1. CHAPTER 1 Genetic Mapping
    2. UNIT 1.1 Collection of Clinical and Epidemiological Data for Genetic Linkage and Association Studies
    3. UNIT 1.2 Pedigree Selection and Information Content
    4. UNIT 1.3 Strategies for Genotyping
    5. UNIT 1.4 Analysis of Genetic Linkage Data for Mendelian Traits
    6. UNIT 1.5 Construction of Reference Genetic Maps
    7. UNIT 1.6 Single-Sperm Typing
    8. UNIT 1.7 Use of LINKAGE Programs for Linkage Analysis
    9. UNIT 1.8 Model-Free Tests for Genetic Linkage
    10. UNIT 1.9 Overview of Linkage Analysis in Complex Traits
    11. UNIT 1.10 Identifying Functional Annotation for Noncoding Genomic Sequences
    12. UNIT 1.11 Homozygosity Mapping Using Pooled DNA
    13. UNIT 1.12 Disease Associations and Family-Based Tests
    14. UNIT 1.13 Human Mapping Databases
    15. UNIT 1.14 Analysis of Gene-Gene Interactions
    16. UNIT 1.15 Detecting Gene-Gene Interaction in Linkage Analysis
    17. UNIT 1.16 Informed Consent for Genetic Research
    18. UNIT 1.17 Population-Based Case-Control Association Studies
    19. UNIT 1.18 Calculation and Use of the Hardy-Weinberg Model in Association Studies
    20. UNIT 1.19 Quality Control Procedures for Genome-Wide Association Studies
    21. UNIT 1.20 Strategies for Pathway Analysis from GWAS Data
    22. UNIT 1.21 Using the PhenX Toolkit to Add Standard Measures to a Study
    23. UNIT 1.22 Methods for Detecting and Correcting for Population Stratification
    24. UNIT 1.23 Overview of Admixture Mapping
    25. UNIT 1.24 Methods for Meta-Analysis of Genetic Data
    26. UNIT 1.25 Genotype Imputation in Genome-Wide Association Studies
    27. UNIT 1.26 Identifying Rare Variants Associated with Complex Traits via Sequencing
    28. UNIT 1.27 Analyzing Copy Number Variation Using SNP Array Data: Protocols for Calling CNV and Association Tests
    29. UNIT 1.28 Statistical Methods for Genome-Wide and Sequencing Association Studies of Complex Traits in Related Samples
    30. UNIT 1.29 Genetic Risk Scores
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      UNIT 1.30 Analysis of Heritability Using Genome-Wide Data
  5. Chapter 2 Genotyping
    1. Introduction
    2. UNIT 2.1 Construction of Small-Insert Libraries from Genomic DNA
    3. UNIT 2.2 Construction of Small-Insert Libraries Enriched for Short Tandem Repeat Sequences by Marker Selection
    4. UNIT 2.3 Colony Hybridization to Screen for Microsatellites
    5. UNIT 2.4 Characterization of (CA)n Microsatellite Repeats from Large-Insert Clones
    6. UNIT 2.5 PCR Methods of Genotyping
    7. UNIT 2.6 Genotyping by Ligation Assays
    8. UNIT 2.7 Restriction Fragment Length Polymorphism Analysis
    9. UNIT 2.8 Automated Fluorescent Genotyping
    10. UNIT 2.9 Single Nucleotide Polymorphism Genotyping Using BeadChip Microarrays
    11. UNIT 2.10 Genotyping Using the TaqMan Assay
    12. UNIT 2.11 High-Throughput Genotyping with Primer Extension Fluorescent Polarization Detection
    13. UNIT 2.12 SNP Genotyping Using the Sequenom MassARRAY iPLEX Platform
    14. UNIT 2.13 CNV Analysis Using TaqMan Copy Number Assays
    15. UNIT 2.14 Quality Control for the Illumina HumanExome BeadChip
  6. Chapter 3 Somatic Cell Hybrids
    1. Introduction
    2. UNIT 3.1 Overview of Somatic Cell Hybrid Mapping
    3. UNIT 3.2 Construction of Somatic Cell Hybrids
    4. UNIT 3.3 Construction and Assay of Radiation Hybrids
    5. UNIT 3.4 Statistical Analysis of Radiation Hybrid Data
    6. UNIT 3.5 Use of Commercially Available Radiation Hybrid Panels
    7. UNIT 3.6 Conversion Technology for the Separation of Maternal and Paternal Copies of Any Autosomal Chromosome in Somatic Cell Hybrids
  7. Chapter 4 Cytogenetics
    1. Introduction
    2. UNIT 4.1 Metaphase Chromosome Preparation from Cultured Peripheral Blood Cells
    3. UNIT 4.2 Chromosome Banding Techniques
    4. UNIT 4.3 In Situ Hybridization to Metaphase Chromosomes and Interphase Nuclei
    5. UNIT 4.4 Microscopy and Image Analysis
    6. UNIT 4.5 High-Resolution FISH Analysis
    7. UNIT 4.6 Comparative Genomic Hybridization
    8. UNIT 4.7 Morphology Antibody Chromosome Technique for Determining Phenotype and Genetic Status of the Same Cell
    9. UNIT 4.8 Chromosome Microdissection
    10. UNIT 4.9 Multicolor Fluorescence In Situ Hybridization (FISH) Approaches for Simultaneous Analysis of the Entire Human Genome
    11. UNIT 4.10 Mitotic Chromosome Preparations from Mouse Cells for Karyotyping
    12. UNIT 4.11 Single-Nucleotide Sequence Discrimination In Situ Using Padlock Probes
    13. UNIT 4.12 Principles and Applications of PRINS in Cytogenetics
    14. UNIT 4.13 The BAC Resource: Tools for Array CGH and FISH
    15. UNIT 4.14 Application of Nexus Copy Number Software for CNV Detection and Analysis
    16. UNIT 4.15 A Multifaceted FISH Approach to Study Endogenous RNAs and DNAs in Native Nuclear and Cell Structures
  8. Chapter 5 Strategies for Large-Insert Cloning and Analysis
    1. Introduction
    2. UNIT 5.1 Pulsed-Field Gel Electrophoresis for Long-Range Restriction Mapping
    3. UNIT 5.2 Construction of YAC Libraries with Large Inserts
    4. UNIT 5.3 Construction of Bacteriophage P1 Libraries with Large Inserts
    5. UNIT 5.4 Construction of Chromosome Jumping and Linking Libraries in E. coli
    6. UNIT 5.5 Screening Large-Insert Libraries by PCR
    7. UNIT 5.6 Screening Large-Insert Libraries by Hybridization
    8. UNIT 5.7 Purification and Characterization of YACs Containing Large Inserts
    9. UNIT 5.8 Rescuing YAC-Insert Ends as E. coli Plasmids
    10. UNIT 5.9 Deriving Probes From Large-Insert Clones by PCR Methods
    11. UNIT 5.10 Constructing Contigs from Large-Insert Clones
    12. UNIT 5.11 Generating Subclones from Large-Insert Genomic Clones
    13. UNIT 5.12 Introduction of Large Insert DNA into Mammalian Cells and Embryos
    14. UNIT 5.13 Building Larger YACs by Recombination
    15. UNIT 5.14 Transfer of YAC Clones to New Hosts by Karyogamy-Deficient Mating
    16. UNIT 5.15 Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries
    17. UNIT 5.16 Navigating Public Physical Mapping Databases
    18. UNIT 5.17 Selective Isolation of Mammalian Genes by TAR Cloning
    19. UNIT 5.18 Human Artificial Chromosome Assembly by Transposon-Based Retrofitting of Genomic BACs with Synthetic Alpha-Satellite Arrays
    20. UNIT 5.19 Large-Scale BAC Clone Restriction Digest Fingerprinting
    21. UNIT 5.20 Fosmid Libraries for Genomic Structural Variation Detection
  9. Chapter 6 Identifying Candidate Genes
    1. Introduction
    2. UNIT 6.1 Isolation of Exons from Cloned DNA by Exon Trapping
    3. UNIT 6.2 Identifying Transcribed Sequences in Arrayed Bacteriophage or Cosmid Libraries
    4. UNIT 6.3 Direct Selection of cDNAs Using Genomic Contigs
    5. UNIT 6.4 Identification of Intron/Exon Boundaries in Genomic DNA by Inverse PCR
    6. UNIT 6.5 GrailEXP and Genome Analysis Pipeline for Genome Annotation
    7. UNIT 6.6 Gene Identification: Methods and Considerations
    8. UNIT 6.7 Sequence Databases: Integrated Information Retrieval and Data Submission
    9. UNIT 6.8 Using BLAST for Performing Sequence Alignment
    10. UNIT 6.9 Accessing the Human Genome
    11. UNIT 6.10 Searching NCBI Databases Using Entrez
    12. UNIT 6.11 Disease and Phenotype Data at Ensembl
    13. UNIT 6.12 Discovery of Rare Homozygous Mutations from Studies of Consanguineous Pedigrees
    14. UNIT 6.13 Genome-Scale Sequencing to Identify Genes Involved in Mendelian Disorders
    15. UNIT 6.14 Using VAAST to Identify Disease-Associated Variants in Next-Generation Sequencing Data
    16. UNIT 6.15 In Silico Functional Annotation of Genomic Variation
  10. Chapter 7 Identifying Sequence Variants
    1. Introduction
    2. UNIT 7.1 Amplification of Sequences from Affected Individuals
    3. UNIT 7.2 Detection of Mutations by RNase Cleavage
    4. UNIT 7.3 Mismatch Detection Using Heteroduplex Analysis
    5. UNIT 7.4 Detection of Mutations by Single-Strand Conformation Polymorphism (SSCP) Analysis and SSCP-Hybrid Methods
    6. UNIT 7.5 Detection of Mutations by Denaturing Gradient Gel Electrophoresis
    7. UNIT 7.6 Chemical Cleavage of Heteroduplex DNA to Identify Mutations
    8. UNIT 7.7 Mutation Detection by Cycle Sequencing
    9. UNIT 7.8 Detection of Mutations by Fluorescence-Assisted Mismatch Analysis (FAMA)
    10. UNIT 7.9 Mutation Detection Using Automated Fluorescence-Based Sequencing
    11. UNIT 7.10 DNA Mutation Detection Using Denaturing High-Performance Liquid Chromatography (DHPLC)
    12. UNIT 7.11 Human Mutation Databases
    13. UNIT 7.12 Single-Strand Conformation Polymorphism Analysis Using Capillary Electrophoresis
    14. UNIT 7.13 Sequence Variant Descriptions: HGVS Nomenclature and Mutalyzer
    15. UNIT 7.14 MLPA and MAPH: Sensitive Detection of Deletions and Duplications
    16. UNIT 7.15 Selection of a Platform for Mutation Detection
    17. UNIT 7.16 PolyPhred Analysis Software for Mutation Detection from Fluorescence-Based Sequence Data
    18. UNIT 7.17 An Overview of Custom Array Sequencing
    19. UNIT 7.18 Targeted Sequencing Using Affymetrix CustomSeq Arrays
    20. UNIT 7.19 A Survey of Copy-Number Variation Detection Tools Based on High-Throughput Sequencing Data
    21. UNIT 7.20 Predicting Functional Effect of Human Missense Mutations Using PolyPhen-2
    22. UNIT 7.21 Quantitative Analysis of Copy Number Variants Based on Real-Time LightCycler PCR
    23. UNIT 7.22 Design of Large-Insert Jumping Libraries for Structural Variant Detection Using Illumina Sequencing
    24. UNIT 7.23 Using XHMM Software to Detect Copy Number Variation in Whole-Exome Sequencing Data
    25. UNIT 7.24 Digital Droplet PCR: CNV Analysis and Other Applications
    26. UNIT 7.25 Interpreting de novo Variation in Human Disease Using denovolyzeR
  11. Chapter 8 Clinical Cytogenetics
    1. Introduction
    2. UNIT 8.1 Overview of Clinical Cytogenetics
    3. UNIT 8.2 Quality Assurance and Quality Control in Clinical Cytogenetics
    4. UNIT 8.3 Preparation of Chorionic Villus Samples for Metaphase Chromosome Analysis and Chromosomal Microarray Analysis
    5. UNIT 8.4 Preparation, Culture, and Analysis of Amniotic Fluid Samples
    6. UNIT 8.5 Preparation and Culture of Products of Conception and Other Solid Tissues for Chromosome Analysis
    7. UNIT 8.6 Analysis of Sister-Chromatid Exchanges
    8. UNIT 8.7 Diagnosis of Fanconi Anemia by Diepoxybutane Analysis
    9. UNIT 8.8 Preparation of Cells from Formalin-Fixed, Paraffin-Embedded Tissue for Use in Fluorescence In Situ Hybridization (FISH) Experiments
    10. UNIT 8.9 Preparation of Amniocytes for Interphase Fluorescence In Situ Hybridization (FISH)
    11. UNIT 8.10 Diagnosis of Cryptic Chromosomal Syndromes by Fluorescence In Situ Hybridization (FISH)
    12. UNIT 8.11 Molecular Cytogenetic Analysis of Telomere Rearrangements
    13. UNIT 8.12 Oligonucleotide Microarrays for Clinical Diagnosis of Copy Number Variation and Zygosity Status
    14. UNIT 8.13 Use of Affymetrix Arrays in the Diagnosis of Gene Copy-Number Variation
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      UNIT 8.14 Interpretation of Genomic Copy Number Variants Using DECIPHER
    16. UNIT 8.15 Noninvasive Prenatal Testing Using Cell-Free Fetal DNA in Maternal Plasma
    17. UNIT 8.16 Using ClinVar as a Resource to Support Variant Interpretation
    18. UNIT 8.17 Copy-Number Variants Detection by Low-Pass Whole-Genome Sequencing
  12. Chapter 9 Clinical Molecular Genetics
    1. Introduction
    2. UNIT 9.1 Overview of Molecular Genetic Diagnosis
    3. UNIT 9.2 Quality Assurance and Quality Improvement in U.S. Clinical Molecular Genetic Laboratories
    4. UNIT 9.3 Multiplex PCR for Identifying DMD Gene Deletions
    5. UNIT 9.4 Simultaneous Detection of Multiple Point Mutations Using Allele-Specific Oligonucleotides
    6. UNIT 9.5 Molecular Analysis of Fragile X Syndrome
    7. UNIT 9.6 Analysis of Repetitive Regions in Myotonic Dystrophy Type 1 and 2
    8. UNIT 9.7 Nonrandom X Chromosome Inactivation Detection
    9. UNIT 9.8 Amplification-Refractory Mutation System (ARMS) Analysis of Point Mutations
    10. UNIT 9.9 Molecular Analysis of Oxidative Phosphorylation Diseases for Detection of Mitochondrial DNA Mutations
    11. UNIT 9.10 Single-Cell DNA and FISH Analysis for Application to Preimplantation Genetic Diagnosis
    12. UNIT 9.11 Protein Truncation Test
    13. UNIT 9.12 Internet Resources in Medical Genetics
    14. UNIT 9.13 Searching Online Mendelian Inheritance in Man (OMIM) for Information on Genetic Loci Involved in Human Disease
    15. UNIT 9.14 Genotyping of Apolipoprotein E: Comparative Evaluation of Different Protocols
    16. UNIT 9.15 Genetic Tests: Clinical Validity and Clinical Utility
    17. UNIT 9.16 Molecular Diagnosis of Hearing Loss
    18. UNIT 9.17 Bone Marrow Engraftment Studies
    19. UNIT 9.18 Infectious Diseases Testing
    20. UNIT 9.19 Overview of Pharmacogenetics
    21. UNIT 9.20 Robust Dosage PCR (RD-PCR) for Highly Accurate Dosage Analysis
    22. UNIT 9.21 The Application of Computer-Based Tools in Obtaining the Genetic Family History
    23. UNIT 9.22 Whole Genome Sequencing: A Considered Approach to Clinical Implementation
    24. UNIT 9.23 Management of Incidental Findings in Clinical Genomic Sequencing
    25. UNIT 9.24 Analysis and Annotation of Whole-Genome or Whole-Exome Sequencing–Derived Variants for Clinical Diagnosis
    26. UNIT 9.25 Molecular Diagnosis of Duchenne Muscular Dystrophy
    27. UNIT 9.26 Huntington Disease: Molecular Diagnostics Approach
    28. UNIT 9.27 Spinal Muscular Atrophy: Overview of Molecular Diagnostic Approaches
    29. UNIT 9.28 Molecular Diagnosis of Cystic Fibrosis
    30. UNIT 9.29 Molecular Diagnosis of Myotonic Dystrophy
    31. UNIT 9.30 Diagnosis of Spinocerebellar Ataxias Caused by Trinucleotide Repeat Expansions
  13. Chapter 10 Cancer Genetics
    1. Introduction
    2. UNIT 10.1 Overview of Genetic Diagnosis in Cancer
    3. UNIT 10.2 Metaphase Harvest and Cytogenetic Analysis of Malignant Hematological Specimens
    4. UNIT 10.3 Metaphase Harvest and Cytogenetic Analysis of Solid Tumor Cultures
    5. 10.4 Molecular Analysis of Gene Rearrangements and Mutations in Acute Leukemias and Myeloid Neoplasms
    6. UNIT 10.5 Molecular Analysis of Gene Amplification in Tumors
    7. UNIT 10.6 Methylation-Specific PCR
    8. UNIT 10.7 Constructing Tissue Microarrays for Research Use
    9. 10.8 Detecting APC Gene Mutations in Familial Adenomatous Polyposis (FAP)
    10. UNIT 10.9 EGF Receptor Testing for Non-Small Cell Lung Carcinomas
    11. UNIT 10.10 p53 Testing for Li-Fraumeni and Li-Fraumeni-Like Syndromes
    12. UNIT 10.11 COSMIC: High-Resolution Cancer Genetics Using the Catalogue of Somatic Mutations in Cancer
    13. UNIT 10.12 Genetic Testing for Hereditary Nonpolyposis Colorectal Cancer (HNPCC)
    14. UNIT 10.13 Identifying Mutations for MYH-Associated Polyposis
    15. UNIT 10.14 Molecular Analysis of Genetic Markers for Non-Hodgkin Lymphomas
  14. Chapter 11 Transcriptional Profiling
    1. Introduction
    2. UNIT 11.1 Genetic Analyses on DNA Microarrays
    3. UNIT 11.2 Oligonucleotide Arrays for Expression Monitoring
    4. UNIT 11.3 Profiling Human Gene Expression with cDNA Microarrays
    5. UNIT 11.4 Analysis of Expression Data: An Overview
    6. UNIT 11.5 Differential Display of mRNA by PCR
    7. UNIT 11.6 One-Step Enzymatic Purification of PCR Products for Direct Sequencing
    8. UNIT 11.7 Serial Analysis of Gene Expression (SAGE): Experimental Method and Data Analysis
    9. UNIT 11.8 Gene Expression Analysis of a Single or Few Cells
    10. UNIT 11.9 An Overview of Spotfire for Gene-Expression Studies
    11. UNIT 11.10 High-Throughput Quantitative Real-Time PCR
    12. UNIT 11.11 Digital Gene Expression by Tag Sequencing on the Illumina Genome Analyzer
    13. UNIT 11.12 High-Throughput Multiplex Sequencing of miRNA
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      UNIT 11.13 RNA-seq Data: Challenges in and Recommendations for Experimental Design and Analysis
    15. UNIT 11.14 Stranded Whole Transcriptome RNA-Seq for All RNA Types
    16. UNIT 11.15 Probe-Directed Degradation (PDD) for Flexible Removal of Unwanted cDNA Sequences from RNA-Seq Libraries
    17. UNIT 11.16 Alternative Splicing Signatures in RNA-seq Data: Percent Spliced in (PSI)
  15. Chapter 12 Vectors for Gene Therapy
    1. Introduction
    2. UNIT 12.1 Biosafety in Handling Gene Transfer Vectors
    3. UNIT 12.2 Semliki Forest Virus and Sindbis Virus Vectors
    4. UNIT 12.3 Preparation of Adenovirus-Polylysine-DNA Complexes
    5. UNIT 12.4 Adenoviral Vectors
    6. UNIT 12.5 Retroviral Vector Production
    7. UNIT 12.6 Particle-Mediated Gene Delivery In Vivo and In Vitro
    8. UNIT 12.7 Production of Vesicular Stomatitis Virus G Glycoprotein (VSV-G) Pseudotyped Retroviral Vectors
    9. UNIT 12.8 Liposome Vectors for In Vivo Gene Delivery
    10. UNIT 12.9 Production of Recombinant Adeno-Associated Viral Vectors
    11. UNIT 12.10 Production and Titration of Lentiviral Vectors
    12. UNIT 12.11 Construction of Replication-Defective Herpes Simplex Virus Vectors
    13. UNIT 12.12 Gene Delivery Using Helper Virus–Free HSV-1 Amplicon Vectors
    14. UNIT 12.13 Helper-Dependent Adenoviral Vectors
    15. UNIT 12.14 Oncolytic Adenoviruses: Design, Generation, and Experimental Procedures
  16. Chapter 13 Delivery Systems for Gene Therapy
    1. Introduction
    2. UNIT 13.1 Gene Transfer to Arteries
    3. UNIT 13.2 DNA Vaccination
    4. UNIT 13.3 Ex Vivo and In Vivo Gene Delivery to the Brain
    5. UNIT 13.4 Gene Delivery to Muscle
    6. UNIT 13.5 Methods for Cancer Gene Therapy
    7. UNIT 13.6 Development of Molecular Genetic Interventions for HIV Infection
    8. UNIT 13.7 Human Hematopoietic Cell Culture, Transduction, and Analyses
    9. UNIT 13.8 Cancer Vaccines
    10. UNIT 13.9 Gene Delivery to the Airway
    11. UNIT 13.10 Gene Delivery to the Liver
    12. UNIT 13.11 Neural Stem Cell-Mediated Delivery of Oncolytic Adenovirus
  17. Chapter 14 Forensic Genetics
    1. Introduction
    2. UNIT 14.1 Overview of Human Identity Testing and Forensic Genetics
    3. UNIT 14.2 Collecting and Handling Samples for Parentage and Forensics DNA-Based Genetic Testing
    4. UNIT 14.3 Isolation of DNA from Forensic Evidence
    5. UNIT 14.4 Molecular Analysis of Paternity
    6. UNIT 14.5 RFLP Analysis of Forensic DNA Samples with Single-Locus VNTR Genetic Markers
    7. UNIT 14.6 Manual Methods for PCR-Based Forensic DNA Analysis
    8. UNIT 14.7 Molecular Analysis of the Human Mitochondrial DNA Control Region for Forensic Identity Testing
    9. UNIT 14.8 Short Tandem Repeat Analysis for Human Identity Testing
    10. UNIT 14.9 Mitochondrial Genome Interrogation for Forensic Casework and Research Studies
  18. Chapter 15 Model Systems for the Analysis of Human Disease
    1. Introduction
    2. UNIT 15.1 Overview of Model Systems for the Analysis of Human Disease
    3. UNIT 15.2 Use of Mouse Models for the Analysis of Human Disease
    4. UNIT 15.3 Use of Zebrafish Models for the Analysis of Human Disease
    5. UNIT 15.4 ENU Mutagenesis in the Mouse
    6. UNIT 15.5 Use of Chicken Models for the Analysis of Human Disease
    7. UNIT 15.6 Yeast as a Model for Human Disease
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      UNIT 15.7 Mouse Genome Editing Using the CRISPR/Cas System
    9. UNIT 15.8 GONAD: A Novel CRISPR/Cas9 Genome Editing Method that Does Not Require Ex Vivo Handling of Embryos
    10. UNIT 15.9 Engineering Large Animal Species to Model Human Diseases
    11. UNIT 15.10 Pronuclear Injection-Based Targeted Transgenesis
    12. UNIT 15.11 Computational Approach to Measuring Myocyte Disarray in Animal Models of Heart Disease
  19. Chapter 16 Automation and Robotics in Genetic Analysis
    1. Introduction
    2. UNIT 16.1 Sample Preparation
  20. Chapter 17 Biochemical Genetics
    1. UNIT 17 Biochemical Genetics
    2. UNIT 17.1 An Overview of Biochemical Genetics
    3. UNIT 17.2 Chromatographic Analysis of Amino and Organic Acids in Physiological Fluids to Detect Inborn Errors of Metabolism
    4. UNIT 17.3 Quantification of Creatine and Guanidinoacetate Using GC-MS and LC-MS/MS for the Detection of Cerebral Creatine Deficiency Syndromes
    5. UNIT 17.4 Detection of Hypo-N-Glycosylation Using Mass Spectrometry of Transferrin
    6. UNIT 17.5 Diagnosis of Inherited Disorders of Galactose Metabolism
    7. UNIT 17.6 Investigational Methods for Peroxisomal Disorders
    8. UNIT 17.7 Management and Quality Assurance in the Biochemical Genetics Laboratory
    9. UNIT 17.8 Acylcarnitine Analysis by Tandem Mass Spectrometry
    10. UNIT 17.9 Diagnosis of Copper Transport Disorders
    11. UNIT 17.10 Determination of Sialylated and Neutral Oligosaccharides in Urine by Mass Spectrometry
    12. UNIT 17.11 Diagnosing Lysosomal Storage Disorders: Pompe Disease
    13. UNIT 17.12 Quantification of Glycosaminoglycans in Urine by Isotope-Dilution Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry
    14. UNIT 17.13 Diagnosing Lysosomal Storage Disorders: Fabry Disease
    15. UNIT 17.14 Diagnosing Lysosomal Storage Disorders: Mucopolysaccharidosis Type II
    16. UNIT 17.15 Diagnosis of Lysosomal Storage Disorders: Gaucher Disease
    17. UNIT 17.16 Diagnosing Lysosomal Storage Disorders: The GM2 Gangliosidoses
    18. UNIT 17.17 Diagnosing Lysosomal Storage Disorders: Mucopolysaccharidosis Type I
    19. UNIT 17.18 Glycosylation Analysis for Congenital Disorders of Glycosylation
    20. UNIT 17.19 Determination of Activity of the Enzymes Hypoxanthine Phosphoribosyl Transferase (HPRT) and Adenine Phosphoribosyl Transferase (APRT) in Blood Spots on Filter Paper
    21. UNIT 17.20 Porphyria Diagnostics—Part 1: A Brief Overview of the Porphyrias
    22. UNIT 17.21 Quantitative Analysis of Total Plasma Homocysteine by LC-MS/MS
    23. UNIT 17.22 Fabry Disease Biomarkers: Analysis of Urinary Lyso-Gb3 and Seven Related Analogs Using Tandem Mass Spectrometry
    24. UNIT 17.23 Tandem Mass Spectrometry Quantitation of Lyso-Gb3 and Six Related Analogs in Plasma for Fabry Disease Patients
    25. UNIT 17.24 High-Risk Screening of Fabry Disease: Analysis of Fifteen Urinary Methylated and Non-Methylated Gb3 Isoforms Using Tandem Mass Spectrometry
    26. UNIT 17.25 Acylglycine Analysis by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS)
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      17.26 High-Risk Screening for Fabry Disease: Analysis by Tandem Mass Spectrometry of Globotriaosylceramide (Gb3) in Urine Collected on Filter Paper
  21. Chapter 18 High-Throughput Sequencing
    1. Introduction
    2. UNIT 18.1 Methods for Generating Shotgun and Mixed Shotgun/Paired-End Libraries for the 454 DNA Sequencer
    3. UNIT 18.2 Improved Protocols for Illumina Sequencing
    4. UNIT 18.3 Targeted Enrichment of Specific Regions in the Human Genome by Array Hybridization
    5. UNIT 18.4 Targeted Exon Sequencing by In-Solution Hybrid Selection
    6. UNIT 18.5 Using the NCBI Map Viewer to Browse Genomic Sequence Data
    7. UNIT 18.6 The UCSC Genome Browser
    8. UNIT 18.7 Searching for Non-B DNA-Forming Motifs Using nBMST (Non-B DNA Motif Search Tool)
    9. UNIT 18.8 Getting Started with Microbiome Analysis: Sample Acquisition to Bioinformatics
    10. UNIT 18.9 Finding Pathogenic Nucleic Acid Sequences in Next Generation Sequencing Data
    11. UNIT 18.10 Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next-Generation Sequencing
    12. UNIT 18.11 1D Genome Sequencing on the Oxford Nanopore MinION
  22. Chapter 19 Mitochondrial Genetics
    1. Introduction
    2. UNIT 19.1 Analysis of Oxidative Damage by Gene-Specific Quantitative PCR
    3. UNIT 19.2 Histochemical Methods for the Diagnosis of Mitochondrial Diseases
    4. UNIT 19.3 Evaluation of the Mitochondrial Respiratory Chain and Oxidative Phosphorylation System Using Polarography and Spectrophotometric Enzyme Assays
    5. UNIT 19.4 Evaluation of the Mitochondrial Respiratory Chain and Oxidative Phosphorylation System Using Blue Native Gel Electrophoresis
    6. UNIT 19.5 Evaluation of the Mitochondrial Respiratory Chain and Oxidative Phosphorylation System Using Yeast Models of OXPHOS Deficiencies
    7. UNIT 19.6 Analysis of Mitochondrial DNA Point Mutation Heteroplasmy by ARMS Quantitative PCR
    8. UNIT 19.7 Real-Time Quantitative PCR Analysis of Mitochondrial DNA Content
    9. UNIT 19.8 Next Generation Sequencing to Characterize Mitochondrial Genomic DNA Heteroplasmy
  23. Chapter 20 Epigenetics
    1. Introduction
    2. UNIT 20.1 Comprehensive High-Throughput Arrays for Relative Methylation (CHARM)
    3. UNIT 20.2 Analysis of Epigenetic Modifications of DNA in Human Cells
    4. UNIT 20.3 Integrative Analysis of Histone ChIP-seq and RNA-seq Data
    5. UNIT 20.4 Assay for Transposase-Accessible Chromatin Using Sequencing (ATAC-seq) Data Analysis
  24. Chapter 21 iPS Cell Models
    1. UNIT 21.2 Practical Integration-Free Episomal Methods for Generating Human Induced Pluripotent Stem Cells
    2. UNIT 21.3 Chemically Defined Culture and Cardiomyocyte Differentiation of Human Pluripotent Stem Cells
    3. UNIT 21.4 Efficient CRISPR/Cas9-Based Genome Engineering in Human Pluripotent Stem Cells
    4. UNIT 21.5 Efficient Generation of Hypothalamic Neurons from Human Pluripotent Stem Cells
    5. UNIT 21.6 Culturing and Neuronal Differentiation of Human Dental Pulp Stem Cells
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      UNIT 21.7 Human Induced Pluripotent Stem (hiPS) Cells from Urine Samples: A Non-Integrative and Feeder-Free Reprogramming Strategy
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      UNIT 21.8 Highly Expandable Human iPS Cell–Derived Neural Progenitor Cells (NPC) and Neurons for Central Nervous System Disease Modeling and High-Throughput Screening
  25. Appendix 1 Abbreviations and Useful Data
    1. APPENDIX 1A Abbreviations Used in this Manual
    2. APPENDIX 1B Overview of Human Repetitive DNA Sequences
    3. APPENDIX 1C Human and Mouse Gene Nomenclature
    4. APPENDIX 1D Reporting of Diagnostic Cytogenetic Results
  26. Appendix 2 Laboratory Guidelines, Equipment, and Stock Solutions
    1. APPENDIX 2A Laboratory Safety
    2. APPENDIX 2B Centrifuges and Rotors
    3. APPENDIX 2C Standard Laboratory Items
    4. APPENDIX 2D Common Buffers, Media, and Stock Solutions
    5. APPENDIX 2E Automation and Robotics for Genetic Analysis
    6. APPENDIX 2F Testing Hygrometers Used in Cytogenetics Laboratories for Metaphase Preparation
  27. Appendix 3 Commonly Used Techniques
    1. APPENDIX 3A References to Molecular Biology Techniques
    2. APPENDIX 3B Isolation of Genomic DNA from Mammalian Cells
    3. APPENDIX 3C Extraction and Precipitation of DNA
    4. APPENDIX 3D Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
    5. APPENDIX 3E Enzymatic Labeling of DNA
    6. APPENDIX 3F Denaturing Polyacrylamide Gel Electrophoresis
    7. APPENDIX 3G Mammalian Cell Tissue Culture
    8. APPENDIX 3H Establishment of Permanent Cell Lines by Epstein-Barr Virus Transformation
    9. APPENDIX 3I Preparation of DNA from Fixed, Paraffin-Embedded Tissue
    10. APPENDIX 3J Internet Basics for Biologists
    11. APPENDIX 3K Analysis of RNA by Northern Blot Hybridization
    12. APPENDIX 3L Introduction to Basic Mouse Handling Techniques
    13. APPENDIX 3M Basic Statistics
    14. APPENDIX 3N Proper Alignment and Adjustment of the Light Microscope
    15. APPENDIX 3O Methods for Quantitative Creatinine Determination
  28. Appendix 4 Chromosome Karyotyping and Idiograms
    1. APPENDIX 4A Karyotyping
    2. APPENDIX 4B ISCN Standard Idiograms
    3. APPENDIX 4C ISCN Rules for Listing Chromosomal Rearrangements
  29. Appendix 5 Genetic Linkage Reference Maps: Access to Internet-Based Resources
    1. APPENDIX 5 Genetic Linkage Reference Maps: Access to Internet-Based Resources
  30. Appendix 6 Human-Mouse Comparative Maps
    1. APPENDIX 6 Human-Mouse Comparative Maps