Current Protocols in Human Genetics
Table of Contents

Foreword by Francis S. Collins
Preface
Contributors

1 Genetic Mapping

1.0 Introduction to Genetic Mapping 
1.1 Collection of Clinical and Epidemiological Information
1.2 Pedigree Selection and Information Content
1.3 Strategies for Genotype Generation
1.4 Analysis of Genetic Linkage Data for Mendelian Traits
1.5 Construction of Reference Genetic Maps
1.6 Single Sperm Typing 
1.7 Use of LINKAGE Programs for Linkage Analysis
1.8 Model-Free Tests for Genetic Linkage
1.9 Overview of Linkage Analysis in Complex Traits
1.10 Genetic Mapping in the Mouse
1.11 Homozygosity Mappingn Using Pooled DNA
1.12 Disease Associations and Family-Based Tests (Revised, July 2003)
1.13 Human Mapping Databases 
1.14 Analysis of Gene-Gene Interactions

2 Genotyping

2.1 Construction of Small-Insert Libraries from Genomic DNA
2.2 Construction of Small-Insert Libraries Enriched for Short Tandem Repeat Sequences by Marker Selection
2.3 Colony Hybridization to Screen for Microsatellites
2.4 Characterization of (CA)n Microsatellite Repeats from Large-Insert Clones
2.5 PCR Methods of Genotyping
2.6 Genotyping by Ligation Assays
2.7 Restriction Fragment Length Polymorphism Analysis
2.8 Automated Fluorescent Genotyping
2.9 Single-Nucleotide Polymorphism Genotyping Using Microarrays
2.10 High-Throughput Genotyping Using the TaqMan Assay 
2.11 High-Throughput Genotyping with Primer Extension Fluorescent Polarization Detection

2.12 SNP Genotyping using Sequenom MassARRAY 7K Platform

3 Somatic Cell Hybrids

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

4 Cytogenetics

4.1 Chromosome Preparation from Cultured Peripheral Blood Cells
4.2 Chromosome Banding Techniques
4.3 In Situ Hybridization to Metaphase Chromosomes and Interphase Nuclei
4.4 Microscopy and Image Analysis
4.5 High-Resolution FISH Analysis
4.6 Comparative Genomic Hybridization
4.7 Morphology Antibody Chromosome Technique for Determining Phenotype and Genotype of the Same Cell
4.8 Chromosome Microdissection
4.9 Multicolor FISH for Simultaneous Analysis of the Entire Human Genome
4.10 Mitotic Chromosome Preparations from Mouse Cells for Karyotyping
4.11 Single-Nucleotide Sequence Discrimination In Situ Using Padlock Probes
4.12 Principles and Applications of PRINS in Cytogenetics 

5 Large-Insert Cloning and Analysis

5.0 Strategies for Large-Insert Cloning and Analysis
5.1 PFGE for Long-Range Restriction Mapping
5.2 Construction of YAC Libraries with Large Inserts
5.3 Construction of Bacteriophage P1 Libraries with Large Inserts
5.4 Construction of Chromosome Jumping and Linking Libraries in E. coli
5.5 Screening of Large-Insert Libraries by PCR
5.6 Screening Large-Insert Libraries by Hybridization
5.7 Purification and Characterization of Large Inserts
5.8 Rescuing YAC Insert Ends as E. coli Plasmids
5.9 Deriving Probes from Large-Insert Clones by PCR Methods
5.10 Constructing Contigs from Large-Insert Clones
5.11 Generating Subclones from Large-Insert Genomic Clones
5.12 Introduction of Large Insert DNA into Mammalian Cells and Embryos
5.13 Building Larger YACs by Recombination
5.14 Transfer of YAC Clones to New Hosts by Karyogamy-Deficient Mating
5.15 Construction of Bacterial Artificial Chromosome Libraries Using the Modified P1 (PAC) System
5.16 Navigating Public Physical Mapping Databases
5.17 elective Isolation of Mammalian Genes by TAR Cloning 

6 Identifying Candidate Genes in Genomic DNA

6.1 Isolation of Exons from Cloned DNA by Exon Trapping
6.2 Identification of Transcribed Sequences in Arrayed Bacteriophage or Cosmid Libraries
6.3 Direct selection of cDNAs Using Genomic Contigs
6.4 Identification of Intron/Exon Boundaries in Genomic DNA by Inverse PCR
6.5 Gene Discovery and Sequence Annotation in the GRAIL-genQuest Environment
6.6 Gene Identification: Methods and Considerations
6.7 Sequence Databases: Integrated Information Retrieval and Data Submission
6.8 Sequence Similarity Searching Using the BLAST Family of Programs
6.9 Accessing the Human Genome 
6.10 Searching the NCBI Databases Using Entrez

7 Searching Candidate Genes for Mutations

7.1 Amplification of Sequences from Affected Individuals
7.2 Detection of Mutations by RNase Cleavage
7.3 Mismatch Detection Using Heteroduplex Analysis
7.4 Detection of Mutations by SSCP Analysis and SSCP-Hybrid Methods
7.5 Detection of Mutations by Denaturing Gradient Gel Electrophoresis
7.6 Chemical Cleavage of Heteroduplex DNA to Identify Mutations
7.7 Mutation Detection by Cycle Sequencing
7.8 Detection of Mutations by Fluorescence-Assisted Mismatch Analysis (FAMA)
7.9 Heterozygote Detection Using Automated Fluorescence-Based Sequencing 
7.10 DNA Mutation Detection Using DHPLC
7.11 Human Mutation Databases (Revised, April 2003)
7.12 Single-Strand Conformation Polymorphism Analysis Using Capillary Electrophoresis 
7.13 Mutation (New, April 2003)  

8 Clinical Cytogenetics

8.1 Overview of Clinical Cytogenetics
8.2 QA and QC in Clinical Cytogenetics 
8.3 Preparation of Metaphase Spreads from Chorionic Villus Samples
8.4 Preparation, Culture, and Analysis of Amniotic Fluid Samples
8.5 Preparation and Culture of Products of Conception and Other Solid Tissues for Chromosome Analysis
8.6 Analysis of Sister-Chromatid Exhchanges
8.7 Diagnosis of Fanconi Anemia by Diepoxybutane Analysis (Revised, April 2003)
8.8 Determination of Chromosomal Aneuploidy Using Paraffin-Embedded Tissue
8.9 Preparation of Amniocytes for Interphase Fluorescence In Situ Hybridization (FISH)
8.10 Diagnosis of Microdeletion Syndromes by FISH
8.11 Molecular Cytogenetic Analysis of Telomere Rearrangements 

9 Clinical Molecular Genetics

9.1 Overview of Molecular Genetic Diagnosis 
9.2 Quality Assurance and Quality Control in Diagnostic Molecular Biology 
9.3 Multiplex PCR for Identification of Dystrophin Gene Deletions
9.4 Simultaneous Detection of Multiple Point Mutations Using Allele-Specific Oligonucleotides
9.5 Molecular Analysis of Fragile X Syndrome (Revised, July 2003)
9.6 Analysis of Trinucleotide Repeats in Myotonic Dystrophy
9.7 Nonrandom X Chromosome Inactivation 
9.8 Amplification-Refractory Mutation System (ARMS) Analysis of Point Mutations
9.9 Detection of Mitochondrial DNA Mutations
 9.10 Single-Cell DNA and FISH Analysis for Application to Preimplantation Genetic Diagnosis 
9.11 Protein Truncation Test
9.12 Internet Resources in Medical Genetics
9.13 Searching OMIM for Information for Genetic Loci 
9.14 Genotype of Apolipoprotein E: Comparative Evaluation of Different Protocols (New, July 2003)
9.15 Clinical Validity and Clinical Utility of Genetic Tests HTML PDF (Size 74KB)
9.16 Molecular Diagnosis of Hearing Loss HTML PDF (Size 134KB)
9.17 Bone Marrow Engraftment Studies

10 Cancer Genetics

10.1 Overview of Genetic Diagnosis in Cancer
10.2 Metaphase Harvest and Cytogenetic Analysis of Malignant Hematological Specimens 
10.3 Metaphase Harvesting and Cytogenetic Analysis of Solid Tumor Cultures
10.4 Molecular Analysis of DNA Rearrangements in Leukemias and Non-Hodgkin's Lymphomas
10.5 Molecular Analysis of Gene Amplification in Tumors
10.6 Methylation-Specific PCR 
10.7 Constructing Tissue Microarrays for Research Use

11 Bioinformatics/Transcriptional Profiling

11.1 Genetic Analyses on DNA Microarrays
        Internet Basics for Biologists has been revised and renumbered as APPENDIX 3J in Feb. 2000
11.2 Oligonucleotide Arrays for Expression Monitoring
11.3 Profiling Human Gene Expression with cDNA Microarrays
11.4 Analysis of Expression Data: An Overview (New, July 2003)
11.5 Differential Display of mRNA by PCR 
11.6 One-Step Enzymatic Purification of PCR Products for Direct Sequencing 
11.7 Serial Analysis of Gene Expression (SAGE) 

12 Vectors for Gene Therapy

12.1 Biosafety in Handling Gene Transfer Vectors
12.2 Semliki Forest Virus and Sindbis Virus Vectors
12.3 Preparation of Adenovirus-Polylysine-DNA Complexes
12.4 Adenoviral Vectors
12.5 Production of Retroviral Vectors
12.6 Particle-Mediated Gene Delivery In Vivo and In Vitro
12.7 Production of Pseudotype-Retroviral Vectors
12.8 Liposome Vectors for In Vivo Gene
12.9 Production of Recombinant Adeno-Associated Viral Vectors
12.10 Production of High-Titer Lentiviral Vectors
12.11 Construction of Replication-Defective Herpes Simplex Virus Vectors
12.12 Gene Delivery Using Helper Virus-Free HSV-1 Amplicon Vectors
9.15 Clinical Validity and Clinical Utility of Genetic Tests HTML PDF (Size 74KB)
Unit 9.16 Molecular Diagnosis of Hearing Loss HTML PDF (Size 134KB)
Unit 9.17 Bone Marrow Engraftment Studies

13 Delivery Systems for Gene Therapy

13.1 Gene Transfer to Arteries 
13.2 DNA Vaccination (Revised, April 2003)
13.3 Ex Vivo and In Vivo Gene Delivery to the Brain
13.4 Gene Delivery to Muscle 
13.5 Methods for Cancer Gene Therapy
13.6 Development of Molecular Genetic Interventions for HIV Infection
13.7 Human Hematopoietic Cell Culture, Transduction, and Analysis
13.8 Cancer Vaccines
13.9 Gene Delivery to the Airway
13.10 Gene Delivery to the Liver 

14 Forensic Genetics

14.1 Overview of Human Identity Testing and Forensic Genetics
14.2 Collecting and Handling Samples for Parentage and Forensics DNA-Based Genetic Testing
14.3 Isolation of DNA from Forensic Evidence
14.4 Moecular Analysis of Paternity
14.5 RFLP Analysis of Forensic DNA Samples with Single-Locus VNTR Genetic Markers
14.6 Manual Methods for PCR-Based Forensic DNA Analysis
14.7 Molecular Analysis of the Human Mitochondrial DNA Control Region for Forensic Identity Testing 
14.8 Short Tandem Repeat Analysis for Human Identity Testing

15 Model Systems for the Analysis of Human Disease

15.1 COMING SOON!
15.2 Use of Mouse Models for the Analysis of Human Disease 
15.3 Use of Zebrafish Models for the Analysis of Human Disease 
15.4 ENU Mutagenesis in the Mouse
15.5 Use of Chicken Models for the Analysis of Human Disease

Chapter 16 Automation and Robotics for Genetic Analysis
Introduction)
16.1 Sample Preparation

Appendices

A.1 Abbreviations and Useful Data 

1. Abbreviations 
2. Overview of Human Repetitive DNA Sequences
3. Human and Mouse Gene Nomenclature
   
4. Reporting of Diagnostic Cytogenetic Results

1. Laboratory Safety Guidelines 
2. Centrifuges and Rotors 
3. Standard Laboratory Equipment 
4. Common Buffers, Stock Solutions, and Media 
5. Automation and Robotics for Genetic Analysis

1. References to Molecular Biology Techniques 
2. Isolation of Genomic DNA from Mammalian Cells 
3. Extraction and Precipitation of DNA 
4. Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy 
5. Enzymatic Labeling DNA 
6. Denaturing Polyacrylamide Gel Electrophoresis 
7. Techniques for Mammalian Cell Tissue Culture 
8. Establishment of Permanent Cell Lines by Epstein-Barr Virus Transformation 
9. Preparation of DNA from Fixed, Paraffin-Embedded Tissue 
10. Internet Basics for Biologists 
11. Analysis of RNA by Northern Blot Hybridization 
12. Introduction to Basic Mouse Handling Techniques
13. Basic Statistics (New, April 2003)
14. Proper Alignment and Adjustment of the Light Microscope (New, July 2003)

1. Karyotyping 
2. ISCN Standard Idiograms 
3. ISCN Rules for Listing Chromosomal Rearrangements

A.6 Human-Mouse Comparative Maps 

Suppliers Appendix 

Indexes

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