Molecular Biology Summer Course Lecture Topics (Partial List)

• Restriction Enzymes and Ligase Enzymes for Cloning DNA
• DNA Manipulations: Linkers and Adaptors
• Cloning Vectors: Plasmids and Bacteriophage
• Bacteriophage Lambda
• Genetic Selection Techniques and Cloning Strategies
• Genomic Library Construction (Including Size Selection)
• cDNA Library Construction (Including Subtraction Libraries)
• Polymerase Chain Reaction (PCR)
• Reverse Transcriptase PCR (RT-PCR)
• Quantitative PCR
• Gel Electrophoresis
• DNA and RNA Isolation and Purification
• Southern Blot Analysis
• Analysis of Gene Expression
• Northern Blot Analysis
• In situ Hybridization Analysis
• DNA Labeling Methods (Radioactive and Non-radioactive)
• DNA/DNA and DNA/RNA Hybridization
• Chain Termination (Dideoxy) DNA Sequencing
• Thermal Cycle Sequencing
• Automated and Non-radioactive DNA Sequencing
• Computer Analysis of DNA Sequences
• Pulse Field Gel Electrophoresis
• Gene Expression and Regulation in Mammalian Systems
• Cloning in Eukaryotic Cells/Transgenic Animals
• Cloning in Cosmids and YAC Vectors
• Immunoscreening Libraries/Immunological Applications
• Gene Expression/Protein Production in Heterologous Hosts

Note: Some of these topics will be combined in a single lecture, while others will take several lectures.

Molecular Biology Summer Course Laboratory Experiments (Partial List)

Experiment #1: Genomic Cloning and DNA Sequencing

• Construction of a size-selected mouse genomic library in the bacteriophage vector Lambda ZAP Express
• Non-radioactive labeling of a mouse DNA probe using PCR.
• Selection of a specific mouse gene (reverse transcriptase) from this library with the non-radioactive DNA probe.
• PCR subcloning of the mouse gene from the lambda cloning vector to a plasmid cloning vector.
• Isolation of recombinant plasmid DNA (plasmid prep).
• Dideoxy DNA sequencing (thermal cycle and non-radioactive) of the cloned mouse gene.
• Computer analysis of the DNA sequence data.

Experiment #2: Genome Analysis

• Isolate and purify mouse genomic DNA from liver and kidney tissues.
• Amplify the prealbumin gene from total mouse DNA using the polymerase chain reaction (PCR).
• Non-radioactive 3' end labeling of a prealbumin oligonucleotide probe and random prime labeling of the mouse reverse transcriptase gene.
• Southern blot analysis of the mouse prealbumin gene and the reverse transcriptase gene.

Experiment #3: Gene Expression Analysis

• Preparation of mRNA from mouse liver total RNA.
• Amplification of prealbumin mRNA by reverse transcriptase PCR.
• RT-PCR analysis of gene expression.
• Quantitative RT-PCR analysis of gene expression.
• RNase protection assays.
  

Experiment #4: cDNA Cloning and PCR Library Analysis

• Construction of a unidirectional cDNA library from mouse liver mRNA in a lambda bacteriophage expression vector (Uni-ZAP).
• Screening the cDNA library by PCR and immunoscreening.
• Analysis of cDNA insert size and the prealbumin gene by PCR.