MOL 2022: Molecular biology: gene technology and its application

• Subject Convenor: Dr Terry Kwok-Schuelein
• Monash handbook

Topics in this unit include recombinant DNA technology; use of bioinformatic tools to handle biological data generated by DNA and protein sequencing; genetic technology and its application in biotechnology.

Lecturing Staff

Dr Terry Kwok-Schuelein	Prof Phil Bird	Dr Michelle Dunstone
Prof John Hamill	Dr Jackie Cheung	Dr Gerald Murray

Organisation of the unit

MOL2022 consists of 3 lectures per week and one 3 hour practical session per week.

The unit is a collaborative course run across three departments: Microbiology, Biochemistry and Molecular Biology, and Biological Sciences. Students will be exposed to laboratory methods in each of the three departments, and classes will include laboratory exercises, computer exercises tutorials, self-directed exercises, oral presentations and written reports.

Topics covered

Theme A: Recombinant DNA Technology

INTRODUCTION TO MOL2022 AND GENE MANIPULATION

Basic cloning requirements. Cutting and joining DNA fragments; vectors; transformation and selection.
Cloning vectors. Desirable properties of plasmid cloning vehicles; development of natural plasmids as cloning vectors; basic plasmid vectors.

CLONING STRATEGIES, LIBRARY CONSTRUCTION AND SCREENING

Hybridisation. DNA denaturation, hybridisation: liquid and solid phase; labelling of the probe; detection systems; Southern blotting; Northern blotting.
Library construction and screening. Making a gene library, screening by DNA hybridisation, screening by immunological assay, screening by protein activity; cloning DNA sequences that encode eukaryotic proteins; the use of specialised vectors.
Gene mapping. Physical mapping of recombinant plasmids: gel electrophoresis of restriction fragments; mapping and subcloning.

CHEMICAL SYNTHESIS, AMPLIFICATION, SEQUENCING AND MUTAGENESIS OF DNA

Chemical synthesis of DNA. Uses of synthesised oligonucleotides.
Polymerase chain reaction. Basic reaction; amplification; specificity; primers; applications.
DNA sequencing. Basic overview of the dideoxy sequencing procedure; automated DNA sequencing; strategies for sequencing large DNA molecules and genomes.
Directed mutagenesis. PCR based mutagenesis; random mutagenesis.

EXPRESSION OF CLONED GENES

Prokaryotic expression systems. Manipulation of cloned genes to achieve expression of proteins.
Eukaryotic expression systems. Yeast, cultured insect cell and mammalian cell expression systems; their advantages over use of prokaryotic expression systems.

Theme B: Bioinformatics – Accessing and Interpreting Gene Sequences

BIOLOGICAL DATABASES

Overview of the genome project.
Introduction to searching databases and retrieving information. Storage of biological data and key storage sites. The main databases (e.g. for gene sequences, protein sequences and structural data). The integration of multiple databases.

ANNOTATION OF GENOMIC SEQUENCES

How computers can be used to interpret large sequences. Searching for open reading frames (ORFs). Discussion of genes based on codon bias and size of ORF. Searching databases with derived sequences from ORFs.

SEQUENCE ALIGNMENTS

Sequence alignments. Similarity vs identity. Structural homology in proteins. Construction of multiple alignments and their interpretation. Structural implications of amino acid conservation.Using sequences to search the databases : BLAST.

PHYLOGENY

Evolution and its relationship to conserved residues. Conservative and non-conservative substitutions.
Construction of a phylogenetic tree and its interpretation.

PROTEIN STRUCTURE AND MODELING

From protein sequence to structural information. Homology modelling of protein structure and comparison with experimental methods.

Theme C: Applications of Recombinant DNA -Molecular Biotechnology

Approaches for genetic manipulation of microbes, plants and animals.

Manipulation of DNA sequence to alter gene structure (PCR based mutation) Concepts of totipotency in eukaryotes (Fungi, Plants, Animals/vertebrates) Introduction of foreign DNA into genomes by physical methods (microinjection, biolistics, electroporation, liposomes)

Introduction of foreign DNA into genomes of fungi, plants and animals using specialized vectors (bacterial, viral) Copy number determination and inheritance of foreign genesGene targeting vs random insertion of foreign DNA sequences Strategies for down regulation of gene expression (antisense genes, oligonucleotides, gene knockouts) RNA interference via double stranded RNA and targeted mutation using TILLING

Studying expression of foreign genes using reporter genes (GUS, GFP, CAT, Luc)
Examples of use of various 5’, 3’ and intronic sequences to alter and control gene
Expression of transgenes and/or proteins intracellularly/extracellularly Stability and inheritance of foreign gene expression (methylation, co-expression)

Examples of uses of transgenic microbes, fungi and animals.

Therapeutic proteins, vaccines and antibodies Manipulation and synthesis of antibodies, small biologically active molecules and novel industrial bioproducts such as biopolymers and oils/solvents Bioremediation, biotransformation and degradation of xenobiotics

Molecular breeding to improve pest/disease resistance and sustainable production by organisms/species of agricultural and industrial importance (yeast; fish; crop plants; animals)

Gene therapy – applications in medicine (cystic fibrosis; ADA deficiency; possibilities for treatment of cancer)

Release of transgenic organisms, products and patenting/regulatory issues arising from this technology

Ethical and safety issues for the environment and consumers (“GMO debate”)