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CBST-1021 Molecular Biology

Chemical & Biosciences Technology Program

Life Sciences Department

Summary: This course details with both practical and applied aspects of molecular biology and includes examination of DNA recombinant technologies, modification, cloning vectors, nucleic acid characterization and PCR. In the laboratory the students will develop practical skills and continue to maintain professional notebooks with a high degree of accountability.

Prerequisites: College or University level Biochemistry or practical knowledge of nucleic acid structure and basic chemistry. Approval through course instructor.

Learning Outcomes:

Upon successful completion of the course material, the student will be able to comprehend fundamental concepts of molecular biology as described below:

1.   Introduction

•    Definitions 

•     History

2.  Recombinant DNA Technology

•    Basic principles

•     Methods

3.      Restriction Modification of DNA

• Definitions / examples

• Specific enzyme systems

4.      Cloning - Specific Vectors

•  Plasmids

•   Bacteriophage

•   Cosmids

5.      Characterizing DNA

•   Determination of DNA length / Restriction Mapping

•   DNA Sequencing /DNA Libraries

•   Hybridization

•   Forensic Molecular Biology

 6.      Polymerase Chain Reaction

•   General / history

•    Mechanics of PCR / typical methodology

•     Inverse PCR / Anchor PCR / PCR applications

 7.      Molecular Diagnostics of Infectious Diseases

•  Phenotype vs. PCR-based systems

•   Nucleic acid analysis without amplification

 8.      PCR and Diagnostics

•   Types of PCR in diagnostics

•   Other amplification techniques

•    Analysis of amplification products

  9.   Genomics - How to Sequence a Genome  

1. The “Shotgun” approach to genome sequencing

 2. The “Clone Contig” approach

 3. Rapid methods for clone contig assembly

•      Clone fingerprinting

•      Clone Contig assembly by “Sequence Tagged Site" (STS)

 4. Using a map to aid sequence assembly

   10.  Genetic maps

•      Obtained by genetic studies using Mendelian principles

•      Short tandem repeats (STRs, microsatellites)

•      Single nucleotide polymorphisms (SNPs)

    11. Physical maps (of a Genome)

•       direct examination of chromosomal DNA

•       mapping reagent

 12. Post-Genomics - Trying to Understand a Genome Sequence

•       locate all the genes & determine their functions

•       2400 out of 6000 genes of S. cerevisiae are still orphans (no assigned functions)

 13. Identifying the genes in a genome sequence

•       Searching for open reading frames (ORFs)

•       Homology search

•       Determining the function of an unknown gene

 14.  Proteomics - Studies of the Transcriptome and Proteome

1.  Analysis Methods:

•       Hybridization analysis

•       Microarray (cDNA)

•       DNA chips

2.  Studying the Proteome: two-dimensional electrophoresis

 15.  Applications of Gene Cloning and DNA  

1. Production of Protein from cloned genes

•     Special Vectors Examples of promoters, Cassettes and gene fusions

•          Problems resulting from the sequence of the foreign gene

•          Problems caused by E. coli

 2. Production of Recombinant Protein by Eukaryotic Cells

3. Recombinant protein from yeast and filamentous fungi

•          advantages

•          disadvantages

4. Animal cells for recombinant protein production

•       protein production in mammalian cells

•       “Pharming” - recombinant protein from live animals

5. Recombinant proteins from plants

16.  Gene Cloning / DNA Analysis in Medicine

1. Production of Recombinant Pharmaceuticals

•       Recombinant insulin

•       Synthesis of human growth hormones in E. coli

•       Synthesis of Recombinant factor VIII

•       Synthesis of other recombinant human proteins

2. Recombinant Vaccines

•       Producing vaccines as recombinant proteins

•       Live Recombinant Vaccines

3.  Identification of Genes Responsible for Human Diseases

4. Gene Therapy :

•       Germline therapy

•       Somatic cell therapy

5. Gene therapy and cancer

 17.   Gene Cloning in Agriculture  

1.      Directed changes with recombinant DNA techniques

2.      Genetic Addition for Plant Genetic Engineering

3.       Gene Subtraction

4.       Problems with Genetically Engineered Plants

5.       Safety concerns with selectable markers

6.    The possibility of harmful effects on the environment

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Example Historical References:

l“The Polymerase Chain Reaction”, Nancy Smyth Templeton – Diagnostic Molecular Pathology 1(1): 58-72, 1992

l“Molecular Diagnostics of Infectious Diseases”, Yi-Wei Tang et al – Clinical Chemistry 43:11 2021-2038, 1997

 

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