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. Chapter 1 Genetic Mapping
    1. Introduction
    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
  4. 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 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
  5. 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
  6. 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 Genotype 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
  7. 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
  8. 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
  9. Chapter 7 Searching for Mutations
    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 Mutation Nomenclature
    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
  10. 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 Metaphase Spreads from Chorionic Villus Samples
    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
    14. Unit 8.13 Use of Affymetrix Mapping Arrays in the Diagnosis of Gene Copy Number Variation
    15. Unit 8.14 Interpretation of Genomic Copy Number Variants Using DECIPHER
  11. 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 Detection of Nonrandom X Chromosome Inactivation
    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 Clinical Validity and Clinical Utility of Genetic Tests
    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
  12. 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. Unit 10.4 Molecular Analysis of Gene Rearrangements and Mutations in Acute Leukemias and Myeloproliferative 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. Unit 10.8 Detecting Mutations in the APC Gene 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 The Catalogue of Somatic Mutations in Cancer (COSMIC)
    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
  13. 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
  14. 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 Production of Retroviral Vectors
    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. 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
  16. 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
  17. 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
  18. Chapter 16 Automation and Robotics in Genetic Analysis
    1. Introduction
    2. Unit 16.1 Sample Preparation
  19. 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
  20. 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 the Illumina Genome Analyzer Sequencing System
    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)
  21. 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
  22. Chapter 20 Epigenetics
    1. Introduction
    2. Unit 20.1 Comprehensive High-Throughput Arrays for Relative Methylation (CHARM)
  23. Appendix 1 Abbreviations and Useful Data
    1. 1A Abbreviations Used in this Manual
    2. 1B Overview of Human Repetitive DNA Sequences
    3. 1C Human and Mouse Gene Nomenclature
    4. 1D Reporting of Diagnostic Cytogenetic Results
  24. Appendix 2 Laboratory Guidelines, Equipment, and Stock Solutions
    1. 2A Laboratory Safety
    2. 2B Centrifuges and Rotors
    3. 2C Standard Laboratory Items
    4. 2D Common Buffers, Media, and Stock Solutions
    5. 2E Automation and Robotics for Genetic Analysis
    6. 2F Testing Hygrometers Used in Cytogenetics Laboratories for Metaphase Preparation
  25. Appendix 3 Commonly Used Techniques
    1. 3A References to Molecular Biology Techniques
    2. 3B Isolation of Genomic DNA from Mammalian Cells
    3. 3C Extraction and Precipitation of DNA
    4. 3D Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
    5. 3E Enzymatic Labeling of DNA
    6. 3F Denaturing Polyacrylamide Gel Electrophoresis
    7. 3G Techniques for Mammalian Cell Tissue Culture
    8. 3H Establishment of Permanent Cell Lines by Epstein-Barr Virus Transformation
    9. 3I Preparation of DNA from Fixed, Paraffin-Embedded Tissue
    10. 3J Internet Basics for Biologists
    11. 3K Analysis of RNA by Northern Blot Hybridization
    12. 3L Introduction to Basic Mouse Handling Techniques
    13. 3M Basic Statistics
    14. 3N Proper Alignment and Adjustment of the Light Microscope
  26. Appendix 4 Chromosome Karyotyping and Idiograms
    1. 4A Karyotyping
    2. 4B ISCN Standard Idiograms
    3. 4C ISCN Rules for Listing Chromosomal Rearrangements
  27. Appendix 5 Genetic Linkage Reference Maps: Access to Internet-Based Resources
    1. 5 Genetic Linkage Reference Maps: Access to Internet-Based Resources
  28. Appendix 6 Human-Mouse Comparative Maps
    1. 6 Human-Mouse Comparative Maps
  29. Appendix Suppliers
    1. Selected Suppliers of Reagents and Equipment

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