Current Protocols in Human Genetics

What's New in Current Protocols in Human Genetics
Supplement 50, August 2006

UNIT 9.12 Internet Resources in Medical Genetics(Revised)
UNIT 10.8 Detecting Mutations in the APC Gene in Familial Adenomatous Polyposis (FAP)(New)
UNIT 10.9 EGF Receptor Testing for Non-Small Lung Carcinomas(New)
UNIT 12.1 Biosafety in Handling Gene Transfer Vectors (Revision)

Current Protocols in Human Genetics is the resource for designing and running successful research projects in all branches of human genetics.

Updated every three months in all formats, CPHG is constantly evolving to keep pace with the very latest discoveries and developments. A year of these quarterly updates is included in the initial CPHG purchase price. That's 570 pages of new (43%) and revised (57%) content on average every year since the initial publication of the work in April 1994! Presently four volumes in its looseleaf print version, CPHG...

* features methods in all areas of genetic research, including disease study, expression analysis, clinical genetics, analysis of sequence variants, cytogenetics, gene therapy, forensic genetics, biochemical genetics and more.
* offers basic to sophisticated methods— many of them equally applicable to other eukaryotic organisms, such as the mouse.
* provides valuable appendices, such as "Commonly Used Techniques" which includes molecular biology and cell culture methods, basic mouse handling techniques, basic statistics.

Edited by: Jonathan L. Haines (Vanderbilt University); Bruce R. Korf (University of Alabama); Cynthia C. Morton (Harvard Medical School); Christine E. Seidman (Harvard Medical School); J.G. Seidman (Harvard Medical School); Douglas R. Smith (Agencourt Biosciences); Guest Editors: Anthony Rosenzweig (Beth Israel Deaconess Medical Center & Harvard Medical School); J. Daniel Sharer (University of Alabama); Past Editors: Nicholas C. Dracopoli (Bristol-Myers Squibb); Donald T. Moir (Microbiotix, Inc.)

Series Editor: Liz Miranker


Chapter 1 Genetic Mapping
Introduction
Unit 1.1 Collection of Clinical and Epidemiological Data for Linkage Studies
Unit 1.2 Pedigree Selection and Information Content
Unit 1.3 Strategies for Genotype Generation
Unit 1.4 Analysis of Genetic Linkage Data for Mendelian Traits
Unit 1.5 Construction of Reference Genetic Maps
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 Comparative Bioinformatics for Mouse and Human Genes: Getting Started
Unit 1.11 Homozygosity Mapping Using Pooled DNA
Unit 1.12 Disease Associations and Family-Based Tests
Unit 1.13 Human Mapping Databases
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

Chapter 2 Genotyping
Introduction
Unit 2.1 Construction of Small-Insert Libraries from Genomic DNA
Unit 2.2 Construction of Small-Insert Libraries Enriched for Short Tandem Repeat Sequences by Marker Selection
Unit 2.3 Colony Hybridization to Screen for Microsatellites
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 High-Throughput Genotyping Using the TaqMan Assay
Unit 2.11 High-Throughput Genotyping with Primer Extension Fluorescent Polarization Detection
Unit 2.12 SNP Genotyping using Sequenom MassARRAY 7K Platform

Chapter 3 Somatic Cell Hybrids
Introduction
Unit 3.1 Overview of Somatic Cell Hybrid Mapping
Unit 3.2 Construction of Somatic Cell Hybrids
Unit 3.3 Construction and Assay of Radiation Hybrids
Unit 3.4 Statistical Analysis of Radiation Hybrid Data
Unit 3.5 Use of Commercially Available Radiation Hybrid Panels

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 Genotype of the Same Cell
Unit 4.8 Chromosome Microdissection
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 4.11 Single-Nucleotide Sequence Discrimination In Situ Using Padlock Probes
Unit 4.12 Principles and Applications of PRINS in Cytogenetics
Unit 4.13 The BAC Resource: Tools for Array CGH and FISH

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 5.3 Construction of Bacteriophage P1 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 5.7 Purification and Characterization of YACs Containing Large Inserts
Unit 5.8 Rescuing YAC-Insert Ends as E. coli Plasmids
Unit 5.9 Deriving Probes From Large-Insert Clones by PCR Methods
Unit 5.10 Constructing Contigs from Large-Insert Clones
Unit 5.11 Generating Subclones from Large-Insert Genomic Clones
Unit 5.12 Introduction of Large Insert DNA into Mammalian Cells and Embryos
Unit 5.13 Building Larger YACs by Recombination
Unit 5.14 Transfer of YAC Clones to New Hosts by Karyogamy-Deficient Mating
Unit 5.15 Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries
Unit 5.16 Navigating Public Physical Mapping Databases
Unit 5.17 Selective Isolation of Mammalian Genes by TAR Cloning

Chapter 6 Identifying Candidate Genes in Genomic DNA
Introduction
Unit 6.1 Isolation of Exons from Cloned DNA by Exon Trapping
Unit 6.2 Identifying Transcribed Sequences in Arrayed Bacteriophage or Cosmid Libraries
Unit 6.3 Direct Selection of cDNAs Using Genomic Contigs
Unit 6.4 Identification of Intron/Exon Boundaries in Genomic DNA by Inverse PCR
Unit 6.5 GrailEXP and Genome Analysis Pipeline for Genome Annotation
Unit 6.6 Gene Identification: Methods and Considerations
Unit 6.7 Sequence Databases: Integrated Information Retrieval and Data Submission
Unit 6.8 Sequence Similarity Searching Using the BLAST Family of Programs
Unit 6.9 Accessing the Human Genome
Unit 6.10 Searching the NCBI Databases Using Entrez

Chapter 7 Searching Candidate Genes for Mutations
Introduction
Unit 7.1 Amplification of Sequences from Affected Individuals
Unit 7.2 Detection of Mutations by RNase Cleavage
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 7.5 Detection of Mutations by Denaturing Gradient Gel Electrophoresis
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 Heterozygote 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

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 Metaphase Spreads from Chorionic Villus Samples
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 Determination of Chromosomal Aneuploidy Using Paraffin-Embedded Tissue
Unit 8.9 Preparation of Amniocytes for Interphase Fluorescence In Situ Hybridization (FISH)
Unit 8.10 Diagnosis of Microdeletion Syndromes by Fluorescence in situ Hybridization (FISH)
Unit 8.11 Molecular Cytogenetic Analysis of Telomere Rearrangements

Chapter 9 Clinical Molecular Genetics
Introduction
Unit 9.1 Overview of Molecular Genetic Diagnosis
Unit 9.2 Quality Assurance and Quality Control in Diagnostic Molecular Biology
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 Trinucleotide Repeats in Myotonic Dystrophy
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 for 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

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 DNA Rearrangements in Leukemias and Non-Hodgkin's Lymphomas
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

Chapter 11 Transcriptional Profiling
Introduction
Unit 11.1 Genetic Analyses on DNA Microarrays
Unit 11.2 Oligonucleotide Arrays for Expression Monitoring
Unit 11.3 Profiling Human Gene Expression with cDNA Microarrays
Unit 11.4 Analysis of Expression Data: An Overview
Unit 11.5 Differential Display of mRNA by PCR
Unit 11.6 One-Step Enzymatic Purification of PCR Products for Direct Sequencing
Unit 11.7 Serial Analysis of Gene Expression (SAGE)
Unit 11.8 Gene Expression Analysis of a Single or Few Cells
Unit 11.9 An Overview of Spotfire for Gene-Expression Studies

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 12.3 Preparation of Adenovirus-Polylysine-DNA Complexes
Unit 12.4 Adenoviral Vectors
Unit 12.5 Production of Retroviral Vectors
Unit 12.6 Particle-Mediated Gene Delivery In Vivo and In Vitro
Unit 12.7 Production of Pseudotype-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 of High-Titer 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 13.5 Methods for Cancer Gene Therapy
Unit 13.6 Development of Molecular Genetic Interventions for HIV Infection
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
Unit 16.1 Sample Preparation

Chapter 17 Biochemical Genetics
Introduction
Unit 17.1 An Overview of Biochemical Genetics

Appendix 1 Abbreviations and Useful Data
1A Abbreviations Used in this Manual
1B Overview of Human Repetitive DNA Sequences
1C Human and Mouse Gene Nomenclature
1D Reporting of Diagnostic Cytogenetic Results

Appendix 2 Laboratory Guidelines, Equipment, and Stock Solutions
2A Laboratory Safety
2B Centrifuges and Rotors
2C Standard Laboratory Items
2D Common Buffers, Media, and Stock Solutions
2E Automation and Robotics for Genetic Analysis

Appendix 3 Commonly Used Techniques
3A References to Molecular Biology Techniques
3B Isolation of Genomic DNA from Mammalian Cells
3C Extraction and Precipitation of DNA
3D Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
3E Enzymatic Labeling of DNA
3F Denaturing Polyacrylamide Gel Electrophoresis
3G Techniques for Mammalian Cell Tissue Culture
3H Establishment of Permanent Cell Lines by Epstein-Barr Virus Transformation
3I Preparation of DNA from Fixed, Paraffin-Embedded Tissue
3J Internet Basics for Biologists
3K Analysis of RNA by Northern Blot Hybridization
3L Introduction to Basic Mouse Handling Techniques
3M Basic Statistics
3N Proper Alignment and Adjustment of the Light Microscope

Appendix 4 Chromosome Karyotyping and Idiograms
4A Karyotyping
4B ISCN Standard Idiograms
4C ISCN Rules for Listing Chromosomal Rearrangements

Appendix 5 Genetic Linkage Reference Maps: Access to Internet-Based Resources
5 Genetic Linkage Reference Maps: Access to Internet-Based Resources

Appendix 6 Human-Mouse Comparative Maps
6 Human-Mouse Comparative Maps

Appendix Suppliers
Selected Suppliers of Reagents and Equipment