I. Lecture Objectives
1. A General History of DNA Science
A. Explain what is meant by "molecular biology is a hybrid discipline."
B. Understand the importance of quantum physics to molecular biology.
C. Discuss how the science of molecular biology arose from the structure-function tradition of the biological sciences.
D. Discuss the contributions of William Harvey, Matthias Schleiden,
Theodor Schwann, and Rudolf Virchow to the
development of modern biology with emphasis on the role of the cell in the living process.
E. Explain how the science of molecular biology arose from the need
to understand the process of heredity, and how that
process was explained from the most general to the most specific way.
F. Discuss the process of natural selection as proposed by Charles Darwin
and Alfred Russell Wallace. Why is this concept
important to the science of molecular biology?
G. Relate the structural process of heredity defined by Gregor Mendel
to the chromosomal role discovered by Walter Sutton,
Thomas Hunt Morgan and his students, Barbara McClintock and Harriet Creighton with regards to variation in inherited
H. Discuss the "one gene-one enzyme" hypothesis of George Beadle and
Edward Tatum. How was this theory tested, and
what impact did it have on the way genetic research is now performed?
I. Discuss the work of Frederick Griffith and Oswald Avery et al.
with regards to the phenomenon of bacterial transformation
and the hereditary role of the DNA molecule.
J. Explain why the bacteriophage studies begun by Max Delbruck, Salvador
Luria, Alfred Hershey and Marsha Chase were
vital to the scientific consensus about DNA as the "master molecule" of the cell.
K. What were the major contributions of the following to our understanding
of the structure and function of the DNA molecule:
Linus Pauling, Maurice Wilkins, Rosalind Franklin, Edward Chargaff, Francis Crick, James Watson, Matthew Meselson,
Frank Stahl, and Arthur Kornberg.
L. What is Crick's "central dogma"? How was this confirmed experimentally?
What amendments have been made to the central
2. Basic Tools and Techniques of the Genetic Scientist
A. Compare the process of cloning on the cellular and molecular levels.
B. Explain what restriction endonucleases are, how they work, and how
they can be used in biotechnology. Define the terms
"restriction" and "methylation".
C. Define the term "recombinant DNA".
D. Compare endonucleases and exonucleases. What is meant by "Type I"
"Type II", and "Type III" endonucleases? Which of
these is most commonly utilized in biotechnology, and why?
E. How are restriction endonucleases named? Give several examples of
restriction enzymes and what effect they have on the
F. Explain the process of agarose gel electrophoresis.
H. Discuss how electrophoresis can be used to purify DNA fragments for further study.
I. Why is Escherichia coli so widely used as a target organism for recombinant DNA research?
J. What is a plasmid vector? How are these categorized based on the
regulation of their replication? What is the relationship
between antibiotic resistance and plasmids?
K. What techniques can be utilized to facilitate the uptake of recombined
vectors by bacterial cells? How can one select for
and isolate recombinant microorganisms?
3. Advanced Techniques of the Genetic Scientist
A. Know how is a gemomic library is prepared. How is it screened when looking for an individual gene?
B. Know what vectors are the most commonly used in the production of
a genomic library? What are the advantages of each
C. How are l phage vector-based sequences "packaged" by appropriate E. coli?
D. What is a temperature-sensitive (ts) lysogen?
E. Understand how it is possible for the
DNA molecule to produce such a large number of unique protein types while
itself composed of only four nucleotide bases carrying the genetic code.
F. Be able to describe the process of screening of a genomic library by plaque hybridization and cDNA radioactive probes.
G. Know the process of restriction mapping and subcloning.
H. Explain how a cDNA library is constructed.
I. Explain how monoclonal antibodies are created and utilized in the screening of cDNA libraries.
J. Discuss how oligonucleotide probes are generated and used.
K. Explain how cloned sequences of DNA are interpreted.
L. Compare and contrast Southern and Northern blotting techniques as means of examining the structure of genomic DNA.
4. Gene Regulation in Development
A. Compare methods of regulating protein
types produced within cells:
- Extracellular modification
- Posttranslational modifications
B. Explain how mRNA "half-life" is determined, and what this means to the production of proteins.
C. Discuss the process of mRNA translation and the effect of mutant tRNA such as "suppressor" tRNA.
D. Explain mRNA modification in the nucleus of the eukaryotic cell prior to its transport to the cytoplasm.
E. Discuss the rearrangement of DNA via
the use of transposable genetic elements (transposons), and explain how
modification is responsible for the increased pathogenicity of protists such as Trypanosoma, alternation of
generation in yeasts such as Saccharomyces, and the diversity of antibodies.
F. Define the terms "consensus sequence",
"zinc fingers", and "leucine zippers". What are the functions of
the TATA box and
CAT box as promoter sites for transcription of eukaryotic genes?
G. Explain what "enhancer regions" are, and how they function.
H. Discuss the purpose of heterogenous
nuclear RNA (hnRNA), and how it is prepared as functional mRNA for later
I. Define the term "homeotic genes" and discuss their importance in the somatic development of Drosophila.
J. Discuss the techniques and purpose of transgenic mouse developmental studies.
5. DNA Science and Human Genetics
A. Explain the importance of the Human Genome Project.
B. Discuss the process of chromosome mapping through the use of restriction fragment length polymorphisms (RFLP).
C. Be able to explain how the polymerase
chain reaction (PCR) occurs, and how this can be used in the discovery
"sequence tagged sites" (STSs) used in the sharing of genome data.
D. Compare fixed-field, pulse-field, and field-inversion techniques of electrophoresis.
E. What is the reason for using yeast artificial chromosomes (YAC) in human genomic studies?
F. Discuss the techniques utilized in the sequencing of large DNA fragments.
G. Explain the importance of the parallel
sequencing projects of E.coli, C. elegans, D. melanogaster,
A. thaliana, and Z. mays to human genome research.
H. What are "reverse genetics"?
I. Discuss how linked RFLP markers were
used in the mapping of the gene associated with Huntington's chorea and
J. Explain the process of "chromosome walking".
K. Know how DNA diagnosis will aid in the treatment of human disease.
L. Compare somatic and germ-line gene therapy.
M. Explain the process of DNA fingerprinting and differentiate between single- and multiple-locus probes.
6. Practical Applications of DNA Science
A. Compare selective breeding and plant
biotechnology, and discuss why plant molecular biology lags behind other
B. Describe how Agrobacterium tumefaciens is used as a mechanism for plant transformation.
C. Discuss techniques used by molecular
biologists to examine plant resistance to viruses, pesticides, herbicides,
increase plant nutritional value.
D. Discuss mechanisms of genetic manipulation in livestock.
E. Describe the techniques used in the
development of artificial human insulin, human growth hormone, and tissue
F. Know how recombinant vaccines are produced.
G. Describe the antiviral mechanisms of nucleoside analogs such as acyclovir and azidothymidine (AZT).
H. Discuss how X-ray crystallography and site-directed mutagenesis is used to describe the structure of a protein molecule.
I. Compare the activity of abzymes (antibody-enzymes)
and ribozymes (ribosomes with enzyme activity).
II. Laboratory Objectives
1. Basic Laboratory Techniques
A. Know the units of measurement utilized
in the biotechnology laboratory and be able to convert between different
B. Demonstrate the ability to aspirate and dispense both large and small volumes of fluids with different micropipettors.
C. Perform proper aseptic technique on a continuing basis.
D. Understand and demonstrate proper safety
precautions and disposal techniques when handling biohazardous and chemical
2. Bacterial Culturing Techniques
Demonstrate proper techniques for:
A. preparation of microbiological media.
B. streaking for isolation of bacterial colonies.
C. preparation of transformed bacterial cultures and mid-log suspension cultures.
D. determination of culture growth using spectrophotometry.
3. DNA Restriction Analysis
A. Demonstrate proper technique for handling
and storage of restriction enzymes, DNA and restriction buffers, and
bacteriophage l DNA.
B. Determine DNA concentration required
for a given number of experiments using the formula C1V1
= C2V2 and prepare the
proper dilution for storage.
C. Perform restriction digests for later analysis.
D. Demonstrate the proper technique for preparing and using agarose gels in DNA electrophoresis.
E. Be able to troubleshoot agarose gels.
F. Determine sensitivity of DNA detection using ethidium bromide and l DNA.
G. Develop restriction maps of circular l DNA for EcoR1 and BamH1 enzymes.
4. DNA Methylation and Enzyme Restriction
A. Prepare sufficient restriction buffer and S-adenosyl methionine (SAM) for experimentation.
B. Perform methylase experiments and gather data for analysis.
5. Transformation of E. coli with plasmid DNA
A. Demonstrate proper sterile technique.
B. Perform transformation with appropriate outcomes.
C. Properly analyze and evaluate results.
6. Purification and Identification of Plasmid DNA
A. Prepare appropriate miniprep for DNA isolation.
B. Know appropriate buffers for DNA storage.
C. Be able to determine the mass of DNA extracted by miniprep procedures.
7. Restriction Analysis of Plasmid DNA
A. Perform proper miniprep restriction digest, including RNase clean-up of plasmid DNA.
B. Prepare agarose gels and perform electrophoresis.
C. Properly analyze and evaluate results.
8. Recombination of Antibiotic Resistance Genes
A. Perform restriction digests of pAMP
(ampicillin-resistance plasmid) and pKAN (kanamycin resistance plasmid)
with agarose gel electrophoresis.
B. Successfully ligate pAMP and pKAN plasmid fragments.
C. Prepare competent E. coli cells for transformation.
D. Determine transformation efficiency for pAMP and pKAN cells.
9. Replica Plating to Identify Mixed E. coli Populations
A. Prepare and perform multiple replica plating from previous pAMP/pKAN sample plates.
B. Calculate percentages of dual-resistant colonies from each plate.
10. Purification and Identification of Recombinant DNA
A. Perform miniprep extraction of recombined E. coli DNA.
B. Perform restriction enzyme digests of l DNA ladder and miniprep plasmid combinations.
C. Analyze and interpret results.
11. Random Amplified Polymorphism Detection (RAPD) Analysis of Plant DNA
A. Successfully extract DNA from plant tissue.
B. Amplify whole genomic DNA using the polymerase chain reaction (PCR).
C. Perform phylogenetic analysis of the data using RAPD software.
12. Detection of Alu by PCR
A. Successfully extract DNA from human cheek cells.
B. Amplify the Alu sequence within the tissue plasminogen activator gene using PCR.
C. Analyze samples from several different lab members for similarity.
D. Evaluate the technique for forensic applications.
13. DNA Fingerprinting
A. Demonstrate appropriate technique for preparing known and unknown DNA samples for enzyme digestion.
B. Analyze separated fragments and compare to determine identities.
14. Miniblot Identification of Plant Pathogens
A. Prepare serological miniprep for the identification of tobacco mosaic virus (TMV).
B. Use the prepared protocol to screen for TMV in an unknown sample.