Current Protocols in Human Genetics

Table of Contents

• Chapter 1 Genetic Mapping
  • Introduction
  • Unit 1.1 Collection of Clinical and Epidemiological Data for Genetic Linkage and Association Studies
  • Unit 1.2 Pedigree Selection and Information Content
  • Unit 1.3 Strategies for Genotyping
  • Unit 1.4 Analysis of Genetic Linkage Data for Mendelian Traits
  • Unit 1.6 Single‐Sperm Typing
  • Unit 1.7 Use of LINKAGE Programs for Linkage Analysis
  • Unit 1.8 Model‐Free Tests for Genetic Linkage
  • Unit 1.9 Overview of Linkage Analysis in Complex Traits
  • Unit 1.10 Identifying Functional Annotation for Noncoding Genomic Sequences
  • Unit 1.11 Homozygosity Mapping Using Pooled DNA
  • Unit 1.12 Disease Associations and Family‐Based Tests
  • Unit 1.14 Analysis of Gene‐Gene Interactions
  • Unit 1.15 Detecting Gene‐Gene Interaction in Linkage Analysis
  • Unit 1.16 Informed Consent for Genetic Research
  • Unit 1.17 Population‐Based Case‐Control Association Studies
  • Unit 1.18 Calculation and Use of the Hardy‐Weinberg Model in Association Studies
  • Unit 1.19 Quality Control Procedures for Genome‐Wide Association Studies
  • Unit 1.20 Strategies for Pathway Analysis from GWAS Data
  • Unit 1.21 Using the PhenX Toolkit to Add Standard Measures to a Study
  • Unit 1.22 Methods for Detecting and Correcting for Population Stratification
  • Unit 1.23 Overview of Admixture Mapping
  • Unit 1.24 Methods for Meta‐Analysis of Genetic Data
• Chapter 2 Genotyping
  • Introduction
  • Unit 2.4 Characterization of (CA)n Microsatellite Repeats from Large‐Insert Clones
  • Unit 2.5 PCR Methods of Genotyping
  • Unit 2.6 Genotyping by Ligation Assays
  • Unit 2.7 Restriction Fragment Length Polymorphism Analysis
  • Unit 2.8 Automated Fluorescent Genotyping
  • Unit 2.9 Single‐Nucleotide Polymorphism Genotyping Using Microarrays
  • Unit 2.10 Genotyping Using the TaqMan Assay
  • Unit 2.11 High‐Throughput Genotyping with Primer Extension Fluorescent Polarization Detection
  • Unit 2.12 SNP Genotyping Using the Sequenom MassARRAY iPLEX Platform
  • Unit 2.13 CNV Analysis Using TaqMan Copy Number Assays
• Chapter 3 Somatic Cell Hybrids
  • Introduction
  • Unit 3.6 Conversion Technology for the Separation of Maternal and Paternal Copies of Any Autosomal Chromosome in Somatic Cell Hybrids
• Chapter 4 Cytogenetics
  • Introduction
  • Unit 4.1 Metaphase Chromosome Preparation from Cultured Peripheral Blood Cells
  • Unit 4.2 Chromosome Banding Techniques
  • Unit 4.3 In Situ Hybridization to Metaphase Chromosomes and Interphase Nuclei
  • Unit 4.4 Microscopy and Image Analysis
  • Unit 4.5 High‐Resolution FISH Analysis
  • Unit 4.6 Comparative Genomic Hybridization
  • Unit 4.7 Morphology Antibody Chromosome Technique for Determining Phenotype and Genetic Status of the Same Cell
  • Unit 4.9 Multicolor Fluorescence In Situ Hybridization (FISH) Approaches for Simultaneous Analysis of the Entire Human Genome
  • Unit 4.10 Mitotic Chromosome Preparations from Mouse Cells for Karyotyping
  • Unit 4.13 The BAC Resource: Tools for Array CGH and FISH
  • Unit 4.14 Application of Nexus Copy Number Software for CNV Detection and Analysis
  • Unit 4.15 A Multifaceted FISH Approach to Study Endogenous RNAs and DNAs in Native Nuclear and Cell Structures
• Chapter 5 Strategies for Large‐Insert Cloning and Analysis
  • Introduction
  • Unit 5.1 Pulsed‐Field Gel Electrophoresis for Long‐Range Restriction Mapping
  • Unit 5.2 Construction of YAC Libraries with Large Inserts
  • Unit 5.4 Construction of Chromosome Jumping and Linking Libraries in E. coli
  • Unit 5.5 Screening Large‐Insert Libraries by PCR
  • Unit 5.6 Screening Large‐Insert Libraries by Hybridization
  • Unit 5.12 Introduction of Large Insert DNA into Mammalian Cells and Embryos
  • Unit 5.15 Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries
  • Unit 5.17 Selective Isolation of Mammalian Genes by TAR Cloning
  • Unit 5.18 Human Artificial Chromosome Assembly by Transposon‐Based Retrofitting of Genomic BACs with Synthetic Alpha‐Satellite Arrays
  • Unit 5.19 Large‐Scale BAC Clone Restriction Digest Fingerprinting
  • Unit 5.20 Fosmid Libraries for Genomic Structural Variation Detection
• Chapter 6 Identifying Candidate Genes
  • Introduction
  • Unit 6.6 Gene Identification: Methods and Considerations
  • Unit 6.7 Sequence Databases: Integrated Information Retrieval and Data Submission
  • Unit 6.8 Using BLAST for Performing Sequence Alignment
  • Unit 6.10 Searching NCBI Databases Using Entrez
  • Unit 6.11 Disease and Phenotype Data at Ensembl
  • Unit 6.12 Discovery of Rare Homozygous Mutations from Studies of Consanguineous Pedigrees
• Chapter 7 Searching for Mutations
  • Introduction
  • Unit 7.3 Mismatch Detection Using Heteroduplex Analysis
  • Unit 7.4 Detection of Mutations by Single‐Strand Conformation Polymorphism (SSCP) Analysis and SSCP‐Hybrid Methods
  • Unit 7.6 Chemical Cleavage of Heteroduplex DNA to Identify Mutations
  • Unit 7.7 Mutation Detection by Cycle Sequencing
  • Unit 7.8 Detection of Mutations by Fluorescence‐Assisted Mismatch Analysis (FAMA)
  • Unit 7.9 Mutation Detection Using Automated Fluorescence‐Based Sequencing
  • Unit 7.10 DNA Mutation Detection Using Denaturing High‐Performance Liquid Chromatography (DHPLC)
  • Unit 7.11 Human Mutation Databases
  • Unit 7.12 Single‐Strand Conformation Polymorphism Analysis Using Capillary Electrophoresis
  • Unit 7.13 Mutation Nomenclature
  • Unit 7.14 MLPA and MAPH: Sensitive Detection of Deletions and Duplications
  • Unit 7.15 Selection of a Platform for Mutation Detection
  • Unit 7.16 PolyPhred Analysis Software for Mutation Detection from Fluorescence‐Based Sequence Data
  • Unit 7.17 An Overview of Custom Array Sequencing
  • Unit 7.18 Targeted Sequencing Using Affymetrix CustomSeq Arrays
  • Unit 7.19 A Survey of Copy‐Number Variation Detection Tools Based on High‐Throughput Sequencing Data
  • Unit 7.20 Predicting Functional Effect of Human Missense Mutations Using PolyPhen‐2
• Chapter 8 Clinical Cytogenetics
  • Introduction
  • Unit 8.1 Overview of Clinical Cytogenetics
  • Unit 8.2 Quality Assurance and Quality Control in Clinical Cytogenetics
  • Unit 8.3 Preparation of Chorionic Villus Samples for Metaphase Chromosome Analysis and Chromosomal Microarray Analysis
  • Unit 8.4 Preparation, Culture, and Analysis of Amniotic Fluid Samples
  • Unit 8.5 Preparation and Culture of Products of Conception and Other Solid Tissues for Chromosome Analysis
  • Unit 8.6 Analysis of Sister‐Chromatid Exchanges
  • Unit 8.7 Diagnosis of Fanconi Anemia by Diepoxybutane Analysis
  • Unit 8.8 Preparation of Cells from Formalin‐Fixed, Paraffin‐Embedded Tissue for Use in Fluorescence In Situ Hybridization (FISH) Experiments
  • Unit 8.9 Preparation of Amniocytes for Interphase Fluorescence In Situ Hybridization (FISH)
  • Unit 8.10 Diagnosis of Cryptic Chromosomal Syndromes by Fluorescence In Situ Hybridization (FISH)
  • Unit 8.11 Molecular Cytogenetic Analysis of Telomere Rearrangements
  • Unit 8.12 Oligonucleotide Microarrays for Clinical Diagnosis of Copy Number Variation and Zygosity Status
  • Unit 8.13 Use of Affymetrix Mapping Arrays in the Diagnosis of Gene Copy Number Variation
  • Unit 8.14 Interpretation of Genomic Copy Number Variants Using DECIPHER
• Chapter 9 Clinical Molecular Genetics
  • Introduction
  • Unit 9.1 Overview of Molecular Genetic Diagnosis
  • Unit 9.2 Quality Assurance and Quality Improvement in U.S. Clinical Molecular Genetic Laboratories
  • Unit 9.3 Multiplex PCR for Identifying DMD Gene Deletions
  • Unit 9.4 Simultaneous Detection of Multiple Point Mutations Using Allele‐Specific Oligonucleotides
  • Unit 9.5 Molecular Analysis of Fragile X Syndrome
  • Unit 9.6 Analysis of Repetitive Regions in Myotonic Dystrophy Type 1 and 2
  • Unit 9.7 Detection of Nonrandom X Chromosome Inactivation
  • Unit 9.8 Amplification‐Refractory Mutation System (ARMS) Analysis of Point Mutations
  • Unit 9.9 Molecular Analysis of Oxidative Phosphorylation Diseases for Detection of Mitochondrial DNA Mutations
  • Unit 9.10 Single‐Cell DNA and FISH Analysis for Application to Preimplantation Genetic Diagnosis
  • Unit 9.11 Protein Truncation Test
  • Unit 9.12 Internet Resources in Medical Genetics
  • Unit 9.13 Searching Online Mendelian Inheritance in Man (OMIM) for Information on Genetic Loci Involved in Human Disease
  • Unit 9.14 Genotyping of Apolipoprotein E: Comparative Evaluation of Different Protocols
  • Unit 9.15 Clinical Validity and Clinical Utility of Genetic Tests
  • Unit 9.16 Molecular Diagnosis of Hearing Loss
  • Unit 9.17 Bone Marrow Engraftment Studies
  • Unit 9.18 Infectious Diseases Testing
  • Unit 9.19 Overview of Pharmacogenetics
  • Unit 9.20 Robust Dosage PCR (RD‐PCR) for Highly Accurate Dosage Analysis
  • Unit 9.21 The Application of Computer‐Based Tools in Obtaining the Genetic Family History
  • Unit 9.22 Whole Genome Sequencing: A Considered Approach to Clinical Implementation
  • Unit 9.23 Management of Incidental Findings in Clinical Genomic Sequencing
• Chapter 10 Cancer Genetics
  • Introduction
  • Unit 10.1 Overview of Genetic Diagnosis in Cancer
  • Unit 10.2 Metaphase Harvest and Cytogenetic Analysis of Malignant Hematological Specimens
  • Unit 10.3 Metaphase Harvest and Cytogenetic Analysis of Solid Tumor Cultures
  • Unit 10.4 Molecular Analysis of Gene Rearrangements and Mutations in Acute Leukemias and Myeloproliferative Neoplasms
  • Unit 10.5 Molecular Analysis of Gene Amplification in Tumors
  • Unit 10.6 Methylation‐Specific PCR
  • Unit 10.7 Constructing Tissue Microarrays for Research Use
  • Unit 10.8 Detecting Mutations in the APC Gene in Familial Adenomatous Polyposis (FAP)
  • Unit 10.9 EGF Receptor Testing for Non-Small Cell Lung Carcinomas
  • Unit 10.10 p53 Testing for Li‐Fraumeni and Li‐Fraumeni‐Like Syndromes
  • Unit 10.11 The Catalogue of Somatic Mutations in Cancer (COSMIC)
  • Unit 10.12 Genetic Testing for Hereditary Nonpolyposis Colorectal Cancer (HNPCC)
  • Unit 10.13 Identifying Mutations for MYH‐Associated Polyposis
  • Unit 10.14 Molecular Analysis of Genetic Markers for Non‐Hodgkin Lymphomas
• Chapter 11 Transcriptional Profiling
  • Introduction
  • Unit 11.2 Oligonucleotide Arrays for Expression Monitoring
  • Unit 11.4 Analysis of Expression Data: An Overview
  • Unit 11.6 One‐Step Enzymatic Purification of PCR Products for Direct Sequencing
  • Unit 11.7 Serial Analysis of Gene Expression (SAGE): Experimental Method and Data Analysis
  • Unit 11.8 Gene Expression Analysis of a Single or Few Cells
  • Unit 11.9 An Overview of Spotfire for Gene‐Expression Studies
  • Unit 11.10 High‐Throughput Quantitative Real‐Time PCR
  • Unit 11.11 Digital Gene Expression by Tag Sequencing on the Illumina Genome Analyzer
  • Unit 11.12 High‐Throughput Multiplex Sequencing of miRNA
• Chapter 12 Vectors for Gene Therapy
  • Introduction
  • Unit 12.1 Biosafety in Handling Gene Transfer Vectors
  • Unit 12.2 Semliki Forest Virus and Sindbis Virus Vectors
  • Unit 12.4 Adenoviral Vectors
  • Unit 12.5 Production of Retroviral Vectors
  • Unit 12.7 Production of Vesicular Stomatitis Virus G Glycoprotein (VSV‐G) Pseudotyped Retroviral Vectors
  • Unit 12.8 Liposome Vectors for In Vivo Gene Delivery
  • Unit 12.9 Production of Recombinant Adeno‐Associated Viral Vectors
  • Unit 12.10 Production and Titration of Lentiviral Vectors
  • Unit 12.11 Construction of Replication‐Defective Herpes Simplex Virus Vectors
  • Unit 12.12 Gene Delivery Using Helper Virus–Free HSV‐1 Amplicon Vectors
  • Unit 12.13 Helper‐Dependent Adenoviral Vectors
• Chapter 13 Delivery Systems for Gene Therapy
  • Introduction
  • Unit 13.1 Gene Transfer to Arteries
  • Unit 13.2 DNA Vaccination
  • Unit 13.3 Ex Vivo and In Vivo Gene Delivery to the Brain
  • Unit 13.4 Gene Delivery to Muscle
  • Unit 13.7 Human Hematopoietic Cell Culture, Transduction, and Analyses
  • Unit 13.8 Cancer Vaccines
  • Unit 13.9 Gene Delivery to the Airway
  • Unit 13.10 Gene Delivery to the Liver
• Chapter 14 Forensic Genetics
  • Introduction
  • Unit 14.1 Overview of Human Identity Testing and Forensic Genetics
  • Unit 14.2 Collecting and Handling Samples for Parentage and Forensics DNA‐Based Genetic Testing
  • Unit 14.3 Isolation of DNA from Forensic Evidence
  • Unit 14.4 Molecular Analysis of Paternity
  • Unit 14.5 RFLP Analysis of Forensic DNA Samples with Single‐Locus VNTR Genetic Markers
  • Unit 14.6 Manual Methods for PCR‐Based Forensic DNA Analysis
  • Unit 14.7 Molecular Analysis of the Human Mitochondrial DNA Control Region for Forensic Identity Testing
  • Unit 14.8 Short Tandem Repeat Analysis for Human Identity Testing
• Chapter 15 Model Systems for the Analysis of Human Disease
  • Introduction
  • Unit 15.1 Overview of Model Systems for the Analysis of Human Disease
  • Unit 15.2 Use of Mouse Models for the Analysis of Human Disease
  • Unit 15.3 Use of Zebrafish Models for the Analysis of Human Disease
  • Unit 15.4 ENU Mutagenesis in the Mouse
  • Unit 15.5 Use of Chicken Models for the Analysis of Human Disease
  • Unit 15.6 Yeast as a Model for Human Disease
• Chapter 16 Automation and Robotics in Genetic Analysis
  • Introduction
• Chapter 17 Biochemical Genetics
  • Unit 17.1 An Overview of Biochemical Genetics
  • Unit 17.2 Chromatographic Analysis of Amino and Organic Acids in Physiological Fluids to Detect Inborn Errors of Metabolism
  • Unit 17.3 Quantification of Creatine and Guanidinoacetate Using GC‐MS and LC‐MS/MS for the Detection of Cerebral Creatine Deficiency Syndromes
  • Unit 17.4 Detection of Hypo‐N‐Glycosylation Using Mass Spectrometry of Transferrin
  • Unit 17.5 Diagnosis of Inherited Disorders of Galactose Metabolism
  • Unit 17.6 Investigational Methods for Peroxisomal Disorders
  • Unit 17.7 Management and Quality Assurance in the Biochemical Genetics Laboratory
  • Unit 17.8 Acylcarnitine Analysis by Tandem Mass Spectrometry
  • Unit 17.9 Diagnosis of Copper Transport Disorders
  • Unit 17.10 Determination of Sialylated and Neutral Oligosaccharides in Urine by Mass Spectrometry
  • Unit 17.11 Diagnosing Lysosomal Storage Disorders: Pompe Disease
  • Unit 17.12 Quantification of Glycosaminoglycans in Urine by Isotope‐Dilution Liquid Chromatography‐Electrospray Ionization Tandem Mass Spectrometry
  • Unit 17.13 Diagnosing Lysosomal Storage Disorders: Fabry Disease
• Chapter 18 High‐Throughput Sequencing
  • Introduction
  • Unit 18.1 Methods for Generating Shotgun and Mixed Shotgun/Paired‐End Libraries for the 454 DNA Sequencer
  • Unit 18.2 Improved Protocols for the Illumina Genome Analyzer Sequencing System
  • Unit 18.3 Targeted Enrichment of Specific Regions in the Human Genome by Array Hybridization
  • Unit 18.4 Targeted Exon Sequencing by In‐Solution Hybrid Selection
  • Unit 18.5 Using the NCBI Map Viewer to Browse Genomic Sequence Data
  • Unit 18.6 The UCSC Genome Browser
  • Unit 18.7 Searching for Non‐B DNA‐Forming Motifs Using nBMST (Non‐B DNA Motif Search Tool)
• Chapter 19 Mitochondrial Genetics
  • Introduction
  • Unit 19.1 Analysis of Oxidative Damage by Gene‐Specific Quantitative PCR
  • Unit 19.2 Histochemical Methods for the Diagnosis of Mitochondrial Diseases
  • Unit 19.3 Evaluation of the Mitochondrial Respiratory Chain and Oxidative Phosphorylation System Using Polarography and Spectrophotometric Enzyme Assays
  • Unit 19.4 Evaluation of the Mitochondrial Respiratory Chain and Oxidative Phosphorylation System Using Blue Native Gel Electrophoresis
  • Unit 19.5 Evaluation of the Mitochondrial Respiratory Chain and Oxidative Phosphorylation System Using Yeast Models of OXPHOS Deficiencies
  • Unit 19.6 Analysis of Mitochondrial DNA Point Mutation Heteroplasmy by ARMS Quantitative PCR
  • Unit 19.7 Real‐Time Quantitative PCR Analysis of Mitochondrial DNA Content
  • Unit 19.8 Next Generation Sequencing to Characterize Mitochondrial Genomic DNA Heteroplasmy
• Chapter 20 Epigenetics
  • Introduction
  • Unit 20.1 Comprehensive High‐Throughput Arrays for Relative Methylation (CHARM)
  • Unit 20.2 Analysis of Epigenetic Modifications of DNA in Human Cells
• Appendix 1 Abbreviations and Useful Data
  • Appendix 1B Overview of Human Repetitive DNA Sequences
  • Appendix 1C Human and Mouse Gene Nomenclature
  • Appendix 1D Reporting of Diagnostic Cytogenetic Results
• Appendix 2 Laboratory Guidelines, Equipment, and Stock Solutions
  • Appendix 2A Laboratory Safety
  • Appendix 2B Centrifuges and Rotors
  • Appendix 2C Standard Laboratory Items
  • Appendix 2E Automation and Robotics for Genetic Analysis
  • Appendix 2F Testing Hygrometers Used in Cytogenetics Laboratories for Metaphase Preparation
• Appendix 3 Commonly Used Techniques
  • Appendix 3B Isolation of Genomic DNA from Mammalian Cells
  • Appendix 3C Extraction and Precipitation of DNA
  • Appendix 3D Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
  • Appendix 3E Enzymatic Labeling of DNA
  • Appendix 3F Denaturing Polyacrylamide Gel Electrophoresis
  • Appendix 3G Techniques for Mammalian Cell Tissue Culture
  • Appendix 3I Preparation of DNA from Fixed, Paraffin‐Embedded Tissue
  • Appendix 3K Analysis of RNA by Northern Blot Hybridization
  • Appendix 3L Introduction to Basic Mouse Handling Techniques
  • Appendix 3M Basic Statistics
  • Appendix 3N Proper Alignment and Adjustment of the Light Microscope
• Appendix 4 Chromosome Karyotyping and Idiograms
  • Appendix 4A Karyotyping
  • Appendix 4B ISCN Standard Idiograms
  • Appendix 4C ISCN Rules for Listing Chromosomal Rearrangements

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