Cover
Title Page
Copyright
Dedication
Preface
Reviewers
Acknowledgments
Contents
Section I:
Fundamentals of Nucleic Acid Biochemistry: An Overview
Chapter 1:
DNA
DNA
DNA Structure
Nucleotides
Substituted Nucleotides
Nucleic Acid
DNA Replication
Polymerases
Enzymes That Metabolize DNA
Restriction Enzymes
DNA Ligase
Other DNA Metabolizing Enzymes
Other Nucleases
Helicases
Methyltransferases
Recombination in Sexually Reproducing Organisms
Recombination in Asexual Reproduction
Conjugation
Transduction
Transformation
Plasmids
S T U D Y Q U E S T I O N S
Chapter 2:
RNA
Transcription
Transcription Initiation
Transcription Elongation
Transcription Termination
Types/Structures of RNA
Ribosomal RNA
Messenger RNA
Messenger RNA Processing
Splicing
Small Nuclear RNA
Transfer RNA
Micro RNAs
Small Interfering RNA
Other Small RNAs
RNA Polymerases
Other RNA-Metabolizing Enzymes
Ribonucleases
RNA Helicases
Regulation of Transcription
Regulation of RNA Synthesis at Initiation
Epigenetics
Histone Modification
DNA Modification
Regulation of Gene Expression by MicroRNA
Regulation of Gene Expression by RNAinterference
S T U D Y Q U E S T I O N S
Chapter 3:
Proteins and the Genetic Code
Amino Acids
Genes and the Genetic Code
The Genetic Code
Translation
Amino Acid Charging
Protein Synthesis
S T U D Y Q U E S T I O N S
Section II:
Common Techniques in Molecular Biology
Chapter 4:
Nucleic Acid Extraction Methods
Isolation of DNA
Preparing the Sample
Nucleated Cells in Suspension (Blood and Bone Marrow Aspirates)
Tissue Samples
Microorganisms
Organic Isolation Methods
Inorganic Isolation Methods
Solid-Phase Isolation
Crude Lysis
Proteolytic Lysis of Fixed Material
Extraction with Chelating Resin
Other Rapid Extraction Methods
Isolation of Mitochondrial DNA
Isolation of RNA
Total RNA
Specimen Collection
Extraction of Total RNA
Proteolytic Lysis of Fixed Material
Isolation of polyA (messenger) RNA
Measurement of Nucleic Acid Quality and Quantity
Electrophoresis
Spectrophotometry
Fluorometry
Microfluidics
S T U D Y Q U E S T I O N S
Chapter 5:
Resolution and Detection of Nucleic Acids
Electrophoresis
Gel Systems
Agarose Gels
Pulsed Field Gel Electrophoresis
Polyacrylamide Gels
Capillary Electrophoresis
Buffer Systems
Buffer Additives
Electrophoresis Equipment
Gel Loading
Detection Systems
Nucleic AcidβSpecific Dyes
Silver Stain
S T U D Y Q U E S T I O N S
Chapter 6:
Analysis and Characterization of Nucleic Acids and Proteins
Restriction Enzyme Mapping
Hybridization Technologies
Southern Blots
Restriction Enzyme Cutting and Resolution
Preparation of Resolved DNA for Blotting (Transfer)
Blotting (Transfer)
Northern Blots
Western Blots
Probes
DNA Probes
RNA Probes
Other Nucleic Acid Probe Types
Protein Probes
Probe Labeling
Nucleic Acid Probe Design
Hybridization Conditions, Stringency
Detection Systems
Interpretation of Results
Array-Based Hybridization
Dot/Slot Blots
Genomic Array Technology
Macroarrays
Microarrays
Bead Array Technology
Solution Hybridization
S T U D Y Q U E S T I O N S
Chapter 7:
Nucleic Acid Amplification
Target Amplification
Polymerase Chain Reaction
Basic PCR Procedure
Components of PCR
Control of PCR Contamination
Prevention of Mispriming
PCR Product Cleanup
PCR Modifications
Transcription-Based Amplification Systems
Probe Amplification
Ligase Chain Reaction
Strand Displacement Amplification
QΞ² Replicase
Signal Amplification
Branched DNA Amplification
Hybrid Capture Assays
Cleavage-Based Amplification
Cycling Probe
S T U D Y Q U E S T I O N S
Chapter 8:
Chromosomal Structure and Chromosomal Mutations
Chromosomal Structure and Analysis
Chromosomal Compaction and Histones
Chromosome Morphology
Visualizing Chromosomes
Detection of Genome and Chromosomal Mutations
Karyotyping
Fluorescence In Situ Hybridization
Interphase FISH
Metaphase FISH
S T U D Y Q U E S T I O N S
Chapter 9:
Gene Mutations
Types of Gene Mutations
Detection of Gene Mutations
Biochemical Methods
Immunoassays
High-Performance Liquid Chromatography (HPLC)
Gas Chromatography
Mass Spectrometry
Nucleic Acid Analyses
Hybridization-Based Methods
Single-Strand Conformation Polymorphism
Denaturing Gradient Gel Electrophoresis
Allele-Specific Oligomer Hybridization
Melt Curve Analysis
Inversion Probe Assay
Heteroduplex Analysis
Array Technology
Sequencing (Polymerization)-Based Methods
Sequence-Specific (Primer) PCR
Allelic Discrimination with Fluorogenic Probes
Dideoxy DNA Fingerprinting
Dye Terminator Incorporation
Protein Truncation Test
Enzymatic and Chemical Cleavage Methods
Restriction Fragment Length Polymorphisms
Heteroduplex Analysis with Single-Strand Specific Nucleases
Base Excision Sequence Scanning
Nonisotopic RNase Cleavage Assay
Invader Assay
Chemical Cleavage
Other Methods
Gene Mutation Nomenclature
Gene Names
S T U D Y Q U E S T I O N S
Chapter 10:
DNA Sequencing
Direct Sequencing
Manual Sequencing
Chemical (Maxam-Gilbert) Sequencing
Dideoxy Chain Termination (Sanger) Sequencing
Automated Fluorescent Sequencing
Approaches to Automated Sequencing
Preparation of the Sequencing Ladder
Electrophoresis
Sequence Interpretation
Pyrosequencing
Bisulfite DNA Sequencing
RNA Sequencing
Next Generation Sequencing
Bioinformatics
The Human Genome Project
S T U D Y Q U E S T I O N S
Section III:
Techniques in the Clinical Laboratory
Chapter 11:
DNA Polymorphisms and Human Identification
Types of Polymorphisms
RFLP Typing
Genetic Mapping with RFLPs
RFLP and Parentage Testing
Human Identification Using RFLP
STR Typing by PCR
STR Nomenclature
Gender Identification
Analysis of Test Results
Matching of Profiles
Allelic Frequencies in Paternity Testing
Sibling Tests
Y-STR
Matching with Y-STRs
Bone Marrow Engraftment Testing Using DNA Polymorphisms
Linkage Analysis
Quality Assurance for Surgical Sections Using STR
Single Nucleotide Polymorphisms
The Human Haplotype Mapping (HapMap) Project
Mitochondrial DNA Polymorphisms
S T U D Y Q U E S T I O N S
Chapter 12:
Detection and Identification of Microorganisms
Specimen Collection
Sample Preparation
Quality Control
Selection of Sequence Targets for Detection of Microorganisms
Molecular Detection of Microorganisms
Bacteria
Respiratory Tract Pathogens
Bordetella pertussis
Legionella pneumophila
Mycobacterium tuberculosis
Chlamydophila (Chlamydia) pneumoniae
Mycoplasma pneumoniae
Streptococcus pneumoniae
Urogenital Tract Pathogens
Neisseria gonorrhoeae and Chlamydia trachomatis
Treponema pallidum
Haemophilus ducreyi
Mycoplasma and Ureaplasma spp.
Viruses
Human Immunodeficiency Virus
Determining HIV Viral Load
HIV Genotyping
Herpes Viruses
Herpes Simplex Virus
Epstein-Barr Virus
Cytomegalovirus
Varicella Zoster Virus
Hepatitis C Virus
Human Papillomavirus
Respiratory Viruses
BK/JC Viruses
Fungi
Parasites
Antimicrobial Agents
Resistance to Antimicrobial Agents
Molecular Detection of Resistance
Beta-Lactam Antibiotic Resistance
Glycopeptide Antibiotic Resistance
Antimicrobial Resistance in M.tuberculosis
Molecular Epidemiology
Molecular Strain Typing Methods for Epidemiological Studies
Plasmid Analysis
Pulsed Field Gel Electrophoresis
Restriction Fragment Length Polymorphism Analysis
Arbitrarily Primed PCR
Amplified Fragment Length Polymorphism (AFLP) Assay
Interspersed Repetitive Elements
Internal Transcribed Spacer Elements
spa Typing
Multilocus Sequence Typing
Comparison of Typing Methods
S T U D Y Q U E S T I O N S
Chapter 13:
Molecular Detection of Inherited Diseases
The Molecular Basis of Inherited Diseases
Chromosomal Abnormalities
Patterns of Inheritance in Single-Gene Disorders
Molecular Basis of Single-Gene Disorders
Lysosomal Storage Diseases
Molecular Diagnosis of Single-Gene Disorders
Factor V Leiden
Hemachromatosis
Cystic Fibrosis
Cytochrome P-450
Methylenetetrahydrofolate Reductase
Single-Gene Disorders with Nonclassical Patterns of Inheritance
Mutations in Mitochondrial Genes
Trinucleotide Repeat Expansion Disorders
Fragile X Syndrome
Huntington Disease
Idiopathic Congenital Central Hypoventilation Syndrome
Other Triplet Expansion Disorders
Genomic Imprinting
Array CGH Technology
Multifactorial Inheritance
Limitations to Molecular Testing
S T U D Y Q U E S T I O N S
Chapter 14:
Molecular Oncology
Classification of Neoplasms
Molecular Basis of Cancer
Analytical Targets of Molecular Testing
Gene and Chromosomal Mutations in Solid Tumors
Human Epidermal Growth Factor Receptor 2,HER2/neu/erb-b2 1 (17q21.1)
Epidermal Growth Factor Receptor, EGFR (7p12)
Kirsten Rat Sarcoma Viral Oncogene Homolog,K-ras (12p12),Neuroblastoma ras,N-ras (1p13),Harvey Rat Sarcoma Viral Oncogene Homolog, H-ras (11p15)
Ewing Sarcoma,EWS (22q12)
Synovial Sarcoma Translocation, Chromosome 18βSynovial Sarcoma Breakpoint 1 and 2,SYT-SSX1, SYT-SSX2 t(X;18)(p11.2;q11.2)
Paired Box-Forkhead in Rhabdomyosarcoma,PAX3-FKHR, PAX7-FKHR,t(1;13),t(2;13)
Tumor Protein 53,TP53 (17p13)
Ataxia Telangiectasia Mutated Gene,ATM (11q22)
Breast Cancer 1 Gene,BRCA1 (17q21), and Breast Cancer 2 Gene,BRCA2 (13q12)
Von Hippel-Lindau Gene,VHL (3p26)
V-myc Avian Myelocytomatosis ViralRelated Oncogene,NeuroblastomaDerived,MYCN or n-myc (2p24)
Rearranged During Transfection (RET) Proto-oncogene,(10q11)
Isocitrate Dehydrogenase 1 (IDH1,2q33.3) and Isocitrase Dehydrogenase 2 (IDH2, 15q26.1)
Other Molecular Abnormalities
Microsatellite Instability
Loss of Heterozygosity
Gene Expression Patterns
Tissue of Origin
Molecular Targets in Hematological Malignancies
Gene Rearrangements in Leukemia and Lymphoma
V(D)J Recombination
Immunoglobulin Heavy Chain Gene Rearrangement in B Cells
Immunoglobulin Light Chain Gene Rearrangement in B Cells
T-Cell Receptor Gene Rearrangement
Detection of Clonality
Immunoglobulin Heavy Chain Gene Rearrangements
Immunoglobulin Light Chain Gene Rearrangements
T-Cell Receptor Gene Rearrangements
PCR Amplicon Banding Patterns
Translocations in Hematological Malignancies
Gene Mutations in Hematological Malignancies
Test Panels
Additional Chromosomal Abnormalities
S T U D Y Q U E S T I O N S
Chapter 15:
DNA-Based Tissue Typing
The MHC Locus
HLA Polymorphisms
HLA Nomenclature
Molecular Analysis of the MHC
Serological Analysis
HLA Typing
Screening
Crossmatching
Mixed Leukocyte Culture
Protein Gel Electrophoresis
DNA-Based Typing
Sequence-Specific Oligonucleotide Probe Hybridization
Sequence-Specific PCR
Sequence-Based Typing
Other DNA-Based Methods
Combining Typing Results
HLA Test Discrepancies
Coordination of HLA Test Methods
Additional Recognition Factors
Minor Histocompatibility Antigens
Nonconventional MHC Antigens
Killer Cell Immunoglobulin-like Receptors
MHC Disease Association
Summary of Laboratory Testing
S T U D Y Q U E S T I O N S
Chapter 16:
Quality Assurance and Quality Control in the Molecular Laboratory
Specimen Handling
Criteria for Accepting Specimens
Collection Tubes for Molecular Testing
Precautions in Sample Processing
Holding and Storage Requirements
Test Performance
Controls
Quality Assurance
Instrument Maintenance
Refrigerators and Freezers
Thermal Cyclers
Centrifuges
Power Supplies
Cameras and Detectors
Fume Hoods and Laminar Flow Hoods
Liquid Handling
Calibrations
Reagents
Reagent Categories
Chemical Safety
Proficiency Testing
Documentation of Test Results
Gene Nomenclature
Gene Sequencing Results
Reporting Results
S T U D Y Q U E S T I O N S
Appendix A: Study Question Answers
Appendix B: Case Study Question Answers
Appendix C: Glossary
Index
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