Portland Community College | Portland, Oregon

The techniques and approaches that are used in molecular biology are rapidly evolving, and our students need to be somewhat familiar with a variety of techniques in order to understand and use the adaptations that develop from them. Obtaining and analyzing DNA sequence data is a critical to many, if not all, molecular biology applications. Theoretical and practical aspects of library screening involves some fundamental principles as well as commonly used techniques. For that reason these two areas are designated as key content for this course. Beyond these, the specific techniques to be covered are left largely to the discretion of the in instructor.
It is expected that the instructor will devise one or two projects which allows some integration of the key content areas and the additional techniques in a meaningful way. In this course, there should not be a great deal of time spent in routine subcloning operations, as that was emphasized in the prior course. Rather, projects should emphasize broadening the base of the students experience , both in theory and in practice such that their experience of molecular biology techniques will prepare them for technology that is currently in wide use in the biotechnology community.

1. Working from biological material, produce a selected region of DNA for sequence analysis and/or cloning.
2. Screen a plasmid or phage library
3. Interpret reports of library-based gene identification and cloning in the primary literature
4. Use standard bioinformatics software for DNA analysis, searches and comparisons among DNA and protein sequences.

1. Experimentally obtain and evaluate DNA sequence information
2. Carry out laboratory project (s) assigned. Maintain a laboratory notebook which properly documents all activities carried out in the lab. Present results of laboratory projects orally in an informal group setting
3. Participate in, and contribute to, all class and team discussions and activities.
4. Write all scheduled examinations

COURSE CONTENT
SKILLS

1. Sequencing: theory, procedure and analysis
2. Bioinformatics: accession, comparisons and alignments (blast etc)
3. Library screening; principles, practical considerations, vector options, screening methods
4. Growth and manipulation of bacteriophage

Other topics may include, but are not limited to:

1. RNA purification and analysis
   1. eg: Northern blot
   2. RNA protection
   3. RT-PCR 
2. PCR Techniques and applications:
   1. primer design
   2. multiplex
   3. quantitative
   4. RT-PCR
   5. real time PCR 
3. DNA protein interactions
   1. yeast two-hybrid system
   2. gel mobility shift assays
   3. DNA footprint analysis

THEMES

1. Adaptability of Molecular biology methods
2. "The Search" (for molecular information in the genetic haystack)
3. Innovation
4. Care for Procedure

ISSUES

1. Controls
2. Maintaining laboratory notebook for continuity and detail
3. Communication
4. Adapting to alternative strategies and details, in real time.

CONCEPTS

1. "The Search" (for molecular information in the genetic haystack)
2. Obtaining and using DNA sequence
3. PCR Techniques and applications: primer design, quantitative, RT-PCR real time
4. RNA purification and analysis
5. DNA-protein interactions