Diagnostic Molecular Biology

Cover
Diagnostic Molecular
Biology
Copyright
Dedication
Preface
Acknowledgments
1
Nucleic Acids: DNA and RNA
	DNA/RNA Is Genetic Material
		Transformation in Bacteria
		DNA Is the Genetic Material for Bacteria
		DNA Is the Genetic Material for Bacteriophage
		RNA Is the Genetic Material for Viruses
	The Components of Nucleic Acids
		Nitrogenous Base
		Pentose Sugar
		Phosphate Group
		The Formation of a Nucleotide
	The Structure of DNA Molecules
		Chargaff's Rules
		The Arrangement of Each Subunit in the DNA Structure
		Alternative Forms of DNA Exist
		Various Forms of RNA
	Physical Properties of Nucleic Acids
		DNA Denaturation
		Secondary Structure of DNA
		Tertiary Structure of DNA
		Secondary and Tertiary Structure of RNA
	DNA Topoisomerase Catalytic Mechanisms
		Type I Topoisomerases
		Type II Topoisomerases
		The Cellular Roles of the Various Topoisomerases
	Kinetics of RNA Folding
2
Nucleic Acid-Based Cellular Activities
	DNA Replication
		The DNA Replication Hypothesis
		DNA Replication Follows the Semiconservative Replication Mechanism
	The Process of DNA Replication
		The Overview of Three Different Phases of Replication
		Semidiscontinuous DNA Replication Mechanism
		Eukaryotic DNA Replication
	Proteins Required for DNA Replication in E. coli
		Helicase and Single-Strand DNA Binding Proteins
		DNA Gyrase
		Primase
		DNA Polymerase
	Coupling DNA Replication
		The Replisome
		The Assembly of the Replisome
	Replication Patterns
		Bidirectional Replication
		Rolling Circle Replication
		Termination of Replication
		Replication at the Telomere
		Mitochondrial Replication
	Fidelity of DNA Replication
		Proofreading Mechanism
		Polymerase Exonuclease Activity
	DNA Mutation
		Point Mutation
		Insertion and Deletion
		Tautomeric Shifts
		Mismatch Repair
	DNA Damage
		Base Analogs
		Alkylating Agents
		UV Radiation
		Ionizing Radiation
		Byproducts Damage
	Damage Repair: Single-Strand Damage Repair
		DNA Repair Through Undoing the Damage
		Excision Repair System
		Postreplication Repair and SOS Repair System
	Damage Repair: Double-Strand Break Repair
		Homologous Recombination
		Nonhomologous End Joining (NHEJ)
3
Gene Expression: Transcription of the Genetic Code
	General Features of RNA Synthesis
		Template Strand
		Coding Strand
	Promoters
		Promoter Structure-Consensus Sequences
		Promoter Structure-UP Element
	RNA Polymerase in Escherichia coli
		The Content of the Holoenzyme
		The Role of Sigma (σ) in Transcription
	The Process of Transcription
		Initiation
		Elongation
		Termination
	The Regulation of Prokaryotic Transcription
		Alternative σ Factors
		RNA Polymerase Switching
		Antitermination
		Operons
		Enhancers
		Transcription Attenuation
		Riboswitches
	Transcription in Eukaryotes
		Eukaryotic RNA Polymerase
		Promoter Structure
		RNA Polymerase I and III-Directed Transcription
	The Order of Events in Eukaryotic Transcription Initiation
		The Role of Transcription Factors-General Transcription Factors
		The Role of Transcription Factors-Transcription Activators
		Epigenetic Regulation of Eukaryotic Transcription
	Transcription Elongation Through the Nucleosomal Barrier
		Nucleosome Mobilization
		H2A-H2B Dimer Removal
	Posttranscriptional Modification of mRNA
		The Cap at the 5 End of Eukaryotic mRNA
		Capping and Polyadenylation Processes
		RNA Splicing
	Noncoding RNAs
		The mRNA Degradation Mechanism Directed by siRNA
		The mRNA Degradation Mechanism Directed by miRNA
		Both siRNA and miRNA Can Repress Transcription
4
Gene Expression: Translation of the Genetic Code
	The Structure and Properties of Amino acids
		General Structure of Amino Acids
		Enantiomeric Molecules
	Classifications of Amino Acids
		Nonpolar Amino Acids
		Neutral (Uncharged) but Polar Amino Acids
		Acidic Amino Acids That Have a Net Negative Charge at pH 7.0
		Basic Amino Acids That Have a Net Positive Charge at pH 7.0
		Alternative Classification of Amino Acids
		Amino Acids Can Join Through Peptide Bonds to Form polymers
		Zwitterions
	Protein Structure
		Primary Structure
		Secondary Structure
		Tertiary Structure
		Quaternary Structure
	Folding into Three-Dimensional Protein Structure
		Fibrous Proteins
		Globular Proteins
		Membrane Proteins
		Protein Denaturation
	The Genetic Code
		Ribonucleotide Bases Are Used as Letters in the Genetic Code
		Wobble Rules
	Translational Machinery: Ribosome
		The Components of Ribosomes
		Ribosomal Structure
	Translational Machinery: tRNA
		The Structure of tRNA
		The Secondary Structure of tRNA
		The Active Form of tRNA
	Preparation for the Translation
		Activation of tRNA
		Dissociation of Ribosomes
	Aminoacyl-tRNA Synthetases
		Classification of Aminoacyl-tRNA Synthetase
		Second Genetic Code
		Proofreading Activity of Aminoacyl-tRNA Synthetases
	Prokaryotic Translation
		Translation Initiation
		Translation Elongation
		Translation Termination
		Coupled Translation
	Eukaryotic Translation
		Initiation
		Elongation
		Termination
	Posttranslational Modification Proteins
		Molecular Chaperones
		Ubiquitinylation: A Process of Protein Degradation
		Sumoylation
5
The Genome
	The Eukaryotic Cell Cycle and Chromosome Dynamics
		The Cell Cycle
		Chromosome Dynamics During the Cell Cycle
	The Genome in Bacteria and Viruses
		Bacterial Genome
		The Organization and Dynamics of Bacterial Chromosome
		Nucleoid-Associated Proteins
		Viral Genome
	Eukaryotic Chromosome Structure
		Human Chromosome: An Example of a Eukaryotic Genome
		Assembly of the Nucleosome Structure
		Histone Variants
	Higher Order Chromatin Structure
		30-nm Chromatin Fiber
		Loops and Coils
		Variation in Chromatin Structure: Euchromosome and Heterochromosome
		Mitotic Chromosome and Its Structural Organization
	Unique Animal Chromosomes
		Lampbrush Chromosome
		Polytene Chromosomes
	Specialized DNA Sequences in Eukaryotic Chromosomes
		Origin of Replication
		Centromere
		Telomere
	Complexity and Gene Number
		Gene Number
		Genome Size
		The Genome Size Is Not Necessarily Related to the Gene Number in Eukaryotes
	The Features of Genomic Sequences
		Nonrepetitive DNA
		Moderately Repetitive Sequences: Interspersed Elements-Repetitive Transposed Sequences
		Moderately Repetitive Sequences: Tandem Repeated DNA-VNTRs and STRs
		Highly Repetitive Sequences
		Eukaryotic Genome Carries DNA Sequences That Do Not Encode Functional Genes
	Organelle Genomes
	Genome Evolution
		Introduction of Mutation
		Fixation of Mutation
		Evolutionary Rate
	Mechanisms of Genome Evolution
		Duplication
		De Novo Origination
		Horizontal Gene Transfer
		Gene Recombination
		New Gene Regulatory Systems
		Transposable Elements
	Molecular Evolution of Repetitive Sequences
	Evolution Rate of Repetitive DNA Sequences
	Evolution of the Prokaryotic Genome
		Genome Reduction: The Streamlining Hypothesis
		Genome Reduction: The Muller Ratchet
		The Impact of Genome Reduction on Host-Associated Bacteria
6
Extraction and Purification of Nucleic Acids and Proteins
	The Importance of DNA Extraction
	DNA Extraction From Whole Blood Sample
		Ficoll-Directed Density Gradient Through Centrifugation
		Quick Extraction Through Proteinase K and Phenol
	DNA Extraction From Dry Blood Spots
		Dry Blood Spot Preparation
		Release the DNA From the Dry Blood Spot
	Noninvasive Human DNA Isolation
		Genomic DNA Purification From Hair
		Genomic DNA Purified From Saliva
		Genomic DNA Extraction From Urine Sample
		Comparison of Different Extraction Methods
	DNA Preparation From Microorganisms
		Chemical Methods
		Chelex-100 Method
	Nucleic Acid Purification
		Organic Purification
		Inorganic Purification
	Solid-Phase Nucleic Acid Extraction
		Diatomaceous Earth Extraction
		Affinity Extraction
		Anion-Exchange Extraction
	DNA Preparation From Formalin-Fixed, Paraffin-Embedded Tissues
		The Mechanism of Fixation by Formaldehyde
		Deparaffinization
	RNA Preparation
		Handling RNA
		Effects of Storage Conditions on RNA Quality
	Extraction of RNA
		Guanidinium Thiocyanate Phenol-Chloroform Extraction
		Spin Column Method
		Isolation of Poly(A)+ RNA
		Removal of rRNA
	Protein Preparation
		Gentle Cell Disruption
		Mechanical Rupture
	Cell Lysis Conditions
		Enzymatic Degradation During Cell Lysis
		Lysis Buffer
		Protein Quality Under Different Lysis Conditions
	Preparation of Clean Protein Samples
		Contaminant Removal by Precipitation
		Acetone Precipitation
		TCA Precipitation
		Methanol-Chloroform Precipitation
		Problems Caused by the Precipitation
	Protein Extraction From Archival Formalin-Fixed Paraffin-Embedded Tissues
		Problems Caused by the Formalin in Tissue Extraction
		Possible Solutions for PPFE Sample Extraction: Heat-Induced Antigen Retrieval (AR) for Tissue Extraction
7
Detection and Analysis of Nucleic Acids
	Determining DNA Purity and Yield
		UV Spectrometry
		NanoDrop Spectrometry
		Fluorometric Methods
		Real-Time PCR Method
	Molecular Separation of DNA
		Basic Principles of Electrophoresis
	Agarose Gel Electrophoresis
		The Gel Matrix
		Orientation of the Agarose Gel
		Size of DNA Molecules and the Mobility
		Conformation of DNA Molecules and the Mobility
		Gel Buffer
		DNA Size Tracking Dye and Density Agents
		Visualization of DNA
	Polyacrylamide Gels
		The Chemistry of Polyacrylamide Gel
		The Principle of Polyacrylamide Gel
		Mobility of DNA Molecules in Acrylamide Gel
		Special Application of Polyacrylamide Gel Electrophoresis: Denaturing Gradient Gel Electrophoresis (DGGE)
	Pulsed-Field Gel Electrophoresis for Large DNA Molecules
		The Basic Principle of Pulsed Field
		Types of PFGE
		Application of PFGE
	Capillary Electrophoresis
		Setup of the Capillary Electrophoresis
		Mobility of the Analytes
		Separation of DNA Molecules
8
Quantification and Analysis of Proteins
	Protein Quantitation
		UV Spectroscopy Method: Detecting Proteins With Absorbance at 280nm
		UV Spectroscopy Measurement: Detecting Proteins With Absorbance at 205nm
		Spectrofluorometric Measurement: Detecting Proteins by Intrinsic Fluorescence Emission
	Colorimetric Protein Assay Techniques
		Biuret Reaction
		Lowry Method
		Bradford Method
		Bicinchoninic Acid Assay (BCA) Method
		Standards and Assay Validation
	Fluorescent Dye-Based Assays
		Microplate Detection Method
		Cuvette Detection Method
		The Limitations of Using Fluorescent Dyes
	The Choice of Measurement Format
		Cuvette Measurement Method
		Cuvette Cleaning Method
		Microplate Method
		Preparation of Samples for Measurement
	Molecular Separation of Protein Molecules Through Column Chromatography
		Size Exclusion Chromatography
		Ion-Exchange Chromatography
		Affinity Chromatography
		Hydrophobic Interaction Chromatography
	Molecular Separation Through Electrophoresis
		Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
		Gel Staining After Electrophoresis: Colorimetric Staining Methods
		Gel Staining After Electrophoresis: Fluorescence Staining Method
	Gel-Based Proteomics: Protein Separation
		Two-Dimensional Gel Electrophoresis
		First Dimension Electrophoresis: Conventional Isoelectric Focusing
		First Dimension Electrophoresis: Immobilized pH Gradient (IPG)
		First Dimension Electrophoresis: Nonequilibrium pH Gel Electrophoresis
		Second Dimension SDS-PAGE
		Postelectrophoresis
		The Analysis of 2DE Images
		Modification of 2DE: Two-Dimensional Difference Gel Electrophoresis
	Sample Buffer Constituents for 2DE
		Chaotropic Agents
		Detergents
		Reducing Agents
		Carrier Ampholytes
		Removal of Interfering Contaminants
	Gel Free Proteomics Techniques: Capillary Electrophoresis
		The Principles of Capillary Electrophoresis
		Microchip Electrophoresis
	Gel Free Proteomics Techniques: Liquid Chromatography
		Reversed-Phase HPLC
		Affinity HPLC
		Gel-Permeation HPLC
		Ligand-Exchange HPLC
		Capillary Electrochromatography
		Comparison of Various Chromatographic Methods
	Protein Analysis: Mass Spectrometer
		ESI-MS
		MALDI-TOF
9
Amplification of Nucleic Acids
	Conventional Amplification Technique: The Polymerase Chain Reaction
		Three Steps of a PCR Reaction
		Analysis of PCR Products
	Modified Nucleic Acid Amplification Techniques
		Reverse-Transcriptase Polymerase Chain Reaction
		Nested Polymerase Chain Reaction
		Multiplex Polymerase Chain Reaction
		Digital Polymerase Chain Reaction
	Parameters That Affect PCR
		The Size of the PCR Product
		The Lengths of the Primers
		Annealing Temperature
	Quantitative PCR
		The Comparison Between qPCR and Conventional PCR
		Real-Time PCR Instruments
		Real-Time PCR Detection Chemistry
	DNA Intercalating Dyes for qPCR
		Principles of the Intercalating Dyes
		Comparison Between SYBR Green and EvaGreen
	Fluorophore-Labeled Oligonucleotides for qPCR
		The Principle of Fluorophore-Labeled Oligonucleotides
		Types of Fluorophores Used in qPCR
		Fluorophore-Labeled Oligonucleotides: Primer-Probe Chemistries
		Hairpin Primer-Probes
		Cyclicon Primer-Probes
		Angler Primer-Probes
	Fluorophore-Labeled Oligonucleotides: Probes-Based Chemistry
		Hydrolysis Probes
		Hybridization Probes
	Fluorophore-Labeled Oligonucleotides: Nucleic acid Analogues
		PNAs
		LNA
		ZNA
		Nonnatural Bases: Plexor Primers
	Isothermal Amplification Techniques
		Nucleic Acid Sequence-Based Amplification (NASBA)
		Signal-Mediated Amplification of RNA Technology (SMART)
		Strand Displacement Amplification (SDA)
		Rolling Circle Amplification
		Loop-Mediated Isothermal Amplification (LAMP)
		Isothermal Multiple Displacement Amplification
		Single Primer Isothermal Amplification
		Helicase-Dependent Amplification (HDA)
10
Characterization of Nucleic Acids and Proteins
	Nucleases
		Endonucleases for Cutting DNA: Restriction Endonucleases
		Different Sources of Type II Restriction Endonucleases
		Restriction-Modification System
		The Frequency of Recognition Sequences in a DNA Molecule
		Analysis of Restriction Digested Fragments
	Polymerases
		DNA Polymerase I
		Klenow Fragment
		Taq DNA polymerase
		Reverse Transcriptase
	DNA Modifying Enzymes and Ligases
		DNA Modifying Enzymes
		DNA Ligase
	The Application of Nucleic Acid Hybridization
		Southern Hybridization
		Parameters for Southern Hybridization
		Northern Hybridization
		Restriction Fragment Length Polymorphism (RFLP) Analysis
		Amplified Fragment Length Polymorphism (AFLP)
	Detection Methods
		Probe
		RFLP Probe
		Labeling With a Radioactive Marker
		Labeling With a Nonradioactive Marker
	Array-Based Hybridization
		Dot Blot and Slot Blot Hybridization
		Macroarrays
		Microarrays
		Microarray-Manufacturing Technology
		Sample Processing and Detection
	Western Blot
		Gel Electrophoresis for Protein Separation
		Methods for Transferring Protein onto Membranes
		Types of Membranes
		Detection of Proteins
		Enzyme-Linked Immunosorbent Assay (ELISA)
11
Techniques in Sequencing
	Chain-Termination DNA Sequencing Technique
		Principle of Dideoxynucleotide Procedure
		Procedure
		DNA Polymerase Used in Chain-Termination Sequencing
		Template for Chain-Termination Sequencing
		Automated DNA Sequencing
	First High-Throughput Sequencing: Pyrosequencing
		Principle of Pyrosequencing
		Advantage of Pyrosequencing
	Techniques to Sequence a Genome
		Shotgun Cloning Strategy
		The Clone Contig Approach
	Assembly Approach for Clone Contigs-Clone Fingerprinting Techniques
		Clone Contig Assembly by Chromosome Walking
		Rapid Methods for Clone Contig Assembly
		Clone Contig Assembly by Sequence Tagged Site Content Analysis
	High-Throughput NGS Strategies
		Fundamentals of NGS Platforms
		Massively Parallel Pyrosequencing: Roche/454 Life Sciences
		Sequencing-by-Synthesis: Illumina/Solexa
		Sequencing by Ligation: Applied Biosystems/SOLiD
		Sequencing by Semiconductor Technology: Compact Personal Genome Machine (PGM) Sequencer
	The Third-Generation Sequencer
		The Helicos Single-Molecule Sequencing Device, HeliScope
		Pacific Biosciences
		Life Technologies Method
		Nanopore Sequencing
	NGS Data Analysis
		Conversion of Raw Data to Base Sequences
		Alignment and Assembly
		NGS Accuracy
		NGS Software
		The Outlook for NGS Technologies
12
Genome and Transcriptome Analysis
	Genome-Wide Sampling Sequencing for Genetic Polymorphisms
		GWSS Without Size Selection
		GWSS With Semi-Size Selection
		GWSS With Size Selection
		GWSS With Selective Amplification
		GWSS Challenges
	Genome-Wide Association Studies
		Identification of SNPs
		Integration of Epigenetic Effects
	Transcriptome Sequencing
		Advantages to Using Whole Transcriptome Analysis
		Whole Transcriptome Analysis With Sequencing Methods and Technologies
	Transcriptome Sequencing: Whole Transcriptome Shotgun Sequencing-EST (Expressed Sequence Tag) Sequencing Procedure
		Source of EST Library
		Preparation of EST Library
	Transcriptome Sequencing: Whole Transcriptome Shotgun Sequencing-RNA-seq Procedure
		The Principle and the Procedure
		Comparison Between EST and RNA-seq
		Analysis of RNA-seq
		Challenges of RNA-seq Analysis
	Whole Transcriptome Tag/Target Sequencing With Restriction Digestion-SAGE (Serial Analysis of Gene Expression)
		Increase of the Number of Tags Can Simplify SAGE Analysis Procedures
		Elongation of Tag/Target Sizes in SAGE Analysis
		Problem With Restriction Digestion and the Solution
	Whole Transcriptome Tag/Target Sequencing Without Restriction Digestion
		Transcriptome Profiling of 3-Ends With Enrichment of PolyA+ RNA
		Transcriptome Profiling of 3-Ends With Enrichment of PolyA+ cDNA
		Transcriptome Profiling of 3-Ends With Custom Primers Containing Oligo(dT) at 3 End for Sequencing
		Whole Transcriptome Analysis With Sequencing: Profiling of Nonpolyadenylated RNAs
	Whole Transcriptome Analysis With Sequencing
		Profiling of Circular RNAs
		Profiling of RNA Methylation
		Profiling of 5-Ends With Enrichment of 5G-CAP
	The RNA Structurome
		Transcriptome-Wide RNA Structural Probing In Vivo
		Process for In Vivo RNA Structures Transcriptome-Wide Techniques
	Metabolomics for Genome-Wide Association Studies: Linking the Genome to the Metabolome
		The Principle of Metabolomics
		Genome-Wide Association Studies With Metabolomics (mGWAS)
13
Molecular Diagnosis of Chromosomal Disorders
	Chromosome Morphology
		The Role of Centromere
		Location and Classification of Centromere
	Cytogenetic Methods for Chromosome
		The Karyotype
		The Methods to Prepare the Karyotype
	Conventional Cytogenetics
		The Conventional Approach to Prepare the Karyotype
		Banding Patterns
		Detection of Genome and Chromosomal Mutations
	Molecular Cytogenetics: Fluorescence In Situ Hybridization (FISH)
		The In Situ Hybridization (ISH)
		The Fluorescence In Situ Hybridization (FISH)
		The Metaphase FISH
		Detection Techniques
		Interphase FISH
	FISH Probes
		WCP Probes
		Centromeric Enumeration Probes
		Locus-Specific Identifier (LSI) Probes
	Advances in FISH Technology
		MFISH and SKY
		Multicolor Banding (MCB)
		Comparative Genomic Hybridization (CGH)
		Array CGH
	FISH Applications in Prenatal and Postnatal Diagnostics and Research
		The Advantage of Using FISH in Clinical Diagnosis
		The Limitation of Using FISH in Diagnosis
	Applications of CGH Analysis
		aCGH in Detecting Chromosomal Imbalance
		aCGH in Cancer Detection
		Prenatal Genetic Diagnosis Using aCGH
		Comparison of Different Rapid Prenatal Screening and Diagnosis
		aCGH in Preimplantation Genetic Diagnosis
	Chromosomal Disorders: Numerical Chromosome Abnormalities
		Trisomy 21
		Trisomy 18
		Trisomy 13
		Prader-Willi Syndrome
	Chromosomal Disorders: Sex Chromosome Abnormalities
		Turner Syndrome
		Klinefelter Syndrome
	Chromosomal Disorders: Structural Chromosome Abnormalities
		Structural Variants
		SNP
		Types of Chromosomal Rearrangements
	The Causes of Chromosome Abnormalities
		Errors in Cell Division
		Maternal Age
14
Molecular Diagnosis of Mutation and Inherited Diseases
	Variations That Cause Human Disease
		Single-Nucleotide Variants
		Small Insertions and Deletions
		Structural Variants
		Alterations in Mitochondrial DNA and Hemizygous Genes
		Human Epigenetic Alterations
	Choice of Techniques
		The Application of PCR in Direct Mutation Tests
		Techniques Used in Unknown Mutations
		Techniques Used in Detecting Gene Structural Changes
		Techniques Used for Many Unknown Mutations or an Unknown Gene
		Whole-Genome Analysis Techniques
		Single-Cell Sequencing
	Single-Gene Disorders
		Achondroplasia
		Alpha-1 Antitrypsin Deficiency
		Autosomal Dominant Polycystic Kidney Disease
		Cystic Fibrosis
		Duane's Retraction Syndrome
		Duchenne Muscular Dystrophy
		Fragile X Syndrome
		Hemophilia
		Huntington's Disease
		Marfan Syndrome
		Sickle Cell Disease
		Thalassemia
		Tay-Sachs Disease
	Polygenic Disorders and Gene Clustering
		Alcohol Dependence
		Charcot-Marie-Tooth Disease
		Cri du chat Syndrome
		Cardiovascular Disease (CVD)
	Mitochondria Disorders
	Diseases Caused by Mutations in Mitochondrial Dynamics Machinery
		Dominant Optic Atrophy (DOA)
		Mitochondrial Dynamics in Neurodegenerative Diseases: Parkinson's Disease
15
Molecular Diagnosis of Infectious Diseases
	Detection of Bacterial Pathogens, Fungal Pathogens, and Viruses
		Diagnosis Using NAATs
		Diagnosis With Sequencing
	Detection of Bacteria
		Bartonellae Species
		Borrelia Species
		Chlamydia trachomatis
		Clostridium difficile
		Mycoplasma pneumoniae
		Rickettsia Species
		Staphylococcus aureus
		Streptococcus pneumonia
		Streptococcus pyogenes
		Streptococcus agalactiae
	Molecular Detection and Characterization of Fungal Pathogens
		PCR Probes and PCR Formats
		The Application of Sequencing Techniques
		Molecular Detection of Aspergillus
	Detection of Viruses
		Diagnoses of Human Immunodeficiency Virus Infection
		Molecular Detection of Human Papillomavirus
		Molecular Detection of Hepatitis Viruses
		The Detection of HBV
	Emerging Technology: MALDI-TOF MS for Microbial Species Identification
		The Principle of MALDI-TOF MS in Clinical Analysis
		Sample Preparation
16
Guidance for Molecular Clinical Laboratory
	The Need for Molecular Analysis
		The Request of Molecular Testing
		Workflow of Molecular Test
	Preanalytical Standards
		Reduction of Identification Errors
		Sample Receipt and Handling
		Minimization of Cross-Contamination
		Quality Control Metrics
		Specimen Requirements
		DNA Requirements
		Library Qualification and Quantification Requirements for NGS
		Choice of Analytical Method
	Analytical Phase
		DNA and RNA Extraction
		Storage of DNA and RNA
		Standard Devices for Sample Quality Assessment
		Validation of Comprehensive Molecular Assays
		Performance of Molecular Tests
	Postanalytical Analysis
		Data Analysis
		Understanding of Test Results
		Decision-Making Process
		Molecular Diagnostic Report
	General Administrative Guidelines
		Quality Assurance for Diagnostic Laboratories
		Standardization and Quality Management
		Quality Management (QM) and Quality Assurance (QA)
		Equipment Maintenance
		Safety
		Biological Hazards
		Chemical Hazards
	Accreditation
		External Quality Assessment (EQA) Scheme
		Molecular Diagnosis Checklist
		Monitor of the Quality
	Challenges for Molecular Diagnosis in a Clinical Laboratory
		Tissue Challenges
		Technological Challenges: A Multitude of Different Molecular Tests
		Mutation Detection
Bibliography
Index
	A
	B
	C
	D
	E
	F
	G
	H
	I
	K
	L
	M
	N
	O
	P
	Q
	R
	S
	T
	U
	V
	W
	X
	Z
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