BIO 2064: Molecular Diagnostics

• Course Outline
• Lab Practical (To Be Determined)
• Review of Molecular Biology Concepts
• Specific subject areas to be covered in molecular diagnostics
 BIO 2064:Molecular Diagnostics
Course Outline
Topic

1. Principles and Application of PCR and RT-PCR

 The evolution of PCR to RT-PCR

 The application of PCR and RT-PCR
2. DNA Sequencing

 Methods and Applications in Molecular Diagnostics, Next Generation Sequencing methods

3. Gene Mutations and Mutation Detection

 Types of gene mutation and type of change within the coding sequence
 Possible consequences of gene mutation outside of the coding sequence
 Mutation rates and frequencies
 Consequences of gene mutation on protein’s structure and function
4. DNA Polymorphisms and Human Identification

 Types of molecular markers: RFLP, AFLP, VNTR, STR, SNP, STS

5. Molecular Detection of Inherited Diseases

 Nucleic acid hybridization assays, enzymatic assays, functional assays

6. Application of DNA Microarrays and Proteomics to Disease Diagnosis

 Array hybridization and gene chips

 Application of DNA and protein microarrays
 Advancement of superarray, gene expression profiling, identification of bio -markers
7. Flow Cytometry

 The principles and instrument overview

 Particle sizes comparison and the histograms produced
 Fluorescence system, cell sorting and detection
 Analysis of the plot and diagram
 Threshold, cell isolation for down-stream proceessing
8. Development of Molecular Diagnostics

 Diagnostic systems, sensitivity and limitations

 Automation and alternations for low cost solutions
 Rapid screening and instrumental analysis
9. Quality Assurance and Quality Control in the Molecular Diagnostics Laboratory
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Introduction: Biological Samples for Molecular Diagnostics

• Organ To Tissues: Methods for molecular diagnostics? (Limited)

• Tissues To Cells: Methods for molecular diagnostics?

 Flow Cytometry Method for cell analysis

• Cells To Bio-Molecules: Methods for molecular diagnostics?

 Multiple Diagnostic Tools Available, including the following molecules:

• DNA (Many Diagnostic Tools)

• RNA (Unstable, Conversion To DNA preferred)
• Proteins (Unstable, time consuming to analyze, protein tools available)
• Carbohydrates (Diagnostic tools available, not commonly used)
• Lipids (Diagnostic tools available, not commonly used)
 Molecular Biology: Review
• cDNA
• Primer
• Linker
• Adapter
• cDNA library
• Types of Library
• Gene Expression Analysis
• Protein Expression Analysis
• DNA labeling
• Protein Labeling
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Molecular Diagnostics: Relevant Tools/Technologies

• DNA Methods (For Detection of DNA Polymorphism)

• DNA Methods (For Detection of DNA Mutations)
• DNA Sequencing: At Least 3 methods to sequence genes or human genome
• RNA Methods (Mainly used for gene expression analysis)
• Protein Methods (Mainly used for gene expression analysis and mutations
that change the protein function
• Cell Methods (To analyze normal and abnormal cells based on
physical properties of human cells and protein markers
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Introduction To Flow Cytometry (For Cell Analysis)

• What is Flow Cytometry?
• Components of Flow Cytometry
• How the components work?
• Collection of data through Flow Cytometry
• Applications of Flow Cytometry
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Introduction To DNA sequencing Methods

• Chemical Sequencing
• Dideoxy Sequencing
• Next Generation Sequencing (NGS)
Application of NGS (Examples)
• Whole Genome Sequencing
• Precision Medicine
• GWAS
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Introduction To Microarrays
• Types of Microarray
• How to prepare samples for microarray analysis
• Limitations/Disadvantages of microarray analysis
 Quality Control and Assurance

• Sample collection and identification

• Sample quality and storage conditions
• Processing of clinical samples
• Validation of clinical tests (molecular diagnostics)
 BIO 2064 Lab Practical : Molecular Diagnostics
Week Lab Practical Topics

1 Lab 1 Introduction To Molecular Diagnostics: Primer designs and PCR

amplification of EV71 DNA

2 Lab 2 Real Time PCR diagnostics of EV71

3 Lab 3 Antibody detection of EV 71 capsid protein (VP1)

4 Lab 4 Molecular detection of hyper-methylation status of DNA

5 Lab 5 Detection of Drug Resistant Cancer Cells