Department of Biology Sample

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1 Syllabus BIOTECHNOLOGY Spring 2013 Instructor: Atanu Duttaroy, Professor Tel: Office: Room 336, Just Hall Teaching Assistant: Mr. Subhas Mukherjee Lecture: Room 337, Just Hall Biology Laboratory: Room 337. Texts: Human Molecular Genetics, By Tom Strachan and Andrew P. Read, 1999 (2nd Edition) Willey-Liss Publication, New York. Additional texts to be consulted: Molecular Biology By Robert Waever (2 nd edition). McGraw Hill publication. Principles of Gene Manipulation By Primrose, Twyman, and Old (6 th Edition). Blackwell Publication. Discovering Genomics, Proteomics and Bioinformatics By Campbell, and Heyer. Cold Spring Harbor Laboratory Press. All lecture notes will be provided as PDF files. Credits: 4 (2 hour lecture + 4 hour lab/wk). Prerequisites: BIO 101 and 102, Genetics, Molecular Biology or Molecular Genetics. Course Objectives: The field of molecular biology and genetics continues to change at an accelerated pace. Molecular genetics already forms the forefront of biomedical research because of its application to the diagnosis of disease and the great potential it offers for treatment of the disease. Thus, study of molecular genetics is of major interest to all students in biological science and medicine, as well as to a wide range of biomedical researchers. Currently, the pace of research in this field is extremely rapid with new information being poured out of the laboratories at an unimaginable rate. To cope with this flood of knowledge, students require two major tools: a good understanding of the molecular genetic principles and capability to use modern techniques. I started with the premise that the syllabus for an university course in molecular methods should reflect the major research issues of the new millennium. One major objective of this course is to prepare our graduate and upper level undergraduate students with an in depth understanding of modern molecular genetic techniques, so that they can better compete in an ever growing biotechnology job market. A course in advanced molecular techniques and applications should benefit our graduate program by

2 providing our graduate students with new tools, which they may be able to use in their own research. Laboratory: The laboratory section of this course provides hands on experience in various advanced as well as commonly used recombinant DNA techniques. In order to fit the experimental schedule, lecture schedules might vary. Laboratory safety: Safety is a major concern in this laboratory. Following guidelines are to be followed; (1) don't restart or stop any machine without asking your TA, (2) don't handle chemicals or solutions without wearing protective gloves, (3) don't use the UV-transilluminator without wearing safety goggles or put the plexiglass top on while viewing a gel, (4) don't dispose any bacterial plates in regular garbage, (5) don't throw any sharp object into the garbage, and (6) don t eat or drink in this laboratory. Exams and Grades: One mid terms and one final exam will be conducted. Graduate assignment: In addition to the three examinations, graduate students will review a current research paper where one or more of the techniques discussed in the class were used. This research paper will be evaluated on the basis of (1) clarity of rationale, (2) description of the specific method(s) used, and (3) a brief interpretation of the data obtained. Major learning experience from this course Upon successful completion, the students will be knowledgeable about: Vector systems used for cloning and expression cloning. DNA labeling, RFLP analysis, DNA finger printing, and DNA microarray analysis. Application of PCR assay for mutation detection, automated DNA sequencing method, and in vitro mutagenesis using PCR. Sequence tagged sites (STS) mapping, Expressed sequence tagged sites (EST), Assembly of cloned contigs, exon mapping, gene finding soft wares Serial analysis of gene expression (SAGE), use of GFP to track gene expression, gel retardation assay, Yeast two hybrid system and phage display methods. Genetic Testing methods using SSCP, Protein truncation test, and DNA chips. Embryonic stem cell (ES) cell technology - gene targeting and gene mapping. targeted gene expression system the GAL4-UAS method. Targeted loss of gene function using the FLP-FRT somatic recombination system.

3 Course content 1. Cell based DNA cloning Fundamentals of DNA technology, Principles of DNA cloning, Preparing DNA libraries, Recombinant screening, Vector systems for cloning different DNA fragments. 2. Cloning for Expression Bacterial expression vectors, Inducible expression vectors, vectors to facilitate protein purification, Baculovirus as Eukaryotic expression system. 3. Nucleic acid hybridization assays Preparation of nucleic acid probes. Nick translation, random primed method, end labeling, RNA labeling, Isotopic and non-isotopic labeling and detection. Principles of nucleic acid hybridization. Melting temperature and hybridization stringency, kinetics of DNA reassociation, dot blot assay, Southern blotting, Northern blotting, RFLP analysis using Southern blotting, DNA fingerprinting, In situ hybridization, DNA hybridization in microarray analysis. 4. PCR, DNA sequencing and in vitro mutagenesis PCR a cell free method of DNA cloning Rapidity, sensitivity and robustness of PCR method, Applications of PCR, assay for known mutations, use of degenerate primers, anchored PCR, Real time PCR. DNA sequencing Chain terminator method, fluorescent labeling and automated detection system. In vitro mutagenesis Oligonucleotide mismatch mutagenesis, add-on mutagenesis. 5. Gene expression studies using cultured cell or cell extracts Expression mapping Tissue in situ hybridization, RT-PCR and mrna differential display, Green Fluorescent Protein (GFP) to track gene expression and subcellular localization of proteins. Identification of regulatory sequences: DNAseI footprinting, Gel retardation assay. Protein-Protein interactions Yeast two-hybrid system, phage-display method.

4 6. Creation and application of transgenic animals: ES cell technology Use of transgenic technology, YAC transgenic, inducible promoters. Use of transgenic technology in gene targeting and gene trapping, human disease model in animals. Targeted overexpression/misexpression of genes GAL4-UAS system Targeted loss of gene function from specific tissue FLP-FRT based somatic recombination analysis. The Cre-loxP system. 7. Genomic, Proteomics, and Bioinformatics Performing BLAST search with a sequence of interest. Nucleic acid and Protein alignment. Significance of E-value, Raw Scores, Bit Scores Analysis of Open Reading Frame Applications of Genomics Functional Genomics Techniques- Microarray and Proteomics

5 LABORATORY SCHEDULES EXERCISE 1: BACTERIAL TRANSFORMATION USING THE pglo SYSTEM EXERCISE 2: PURIFICATION OF GFP PROTEIN. EXERCISE3: DNA RESTRICTION DIGESTION AND ANALYSIS. EXERCISE4: DNA FINGERPRINTING. EXERCISE 5: SIZE EXCLUSION CHROMATOGRAPHY EXERCISE6: ANALYSIS OF MUTANT HAEMOGLOBIN THROUGH RFLP ANALYSIS. EXERCISE 8: SOUTHERN HYBRIDIZATION EXERCISE 9: NORTEHRN HYBRIDIZATION EXERCISE 10: WESTERN ANALYSIS

6 GRADING: Lecture Mid term Quiz Lab Reports Final TOTAL 150 points 50 points 50 points 150 points 400 points General Grade Points for: A- 90% and above B- 80% and above C- 70% and above. Incomplete Grades upon request: If your grade is not satisfactory due to unexpected personal reason, please make a written request for an incomplete grade. This will provide another opportunity for you to improve your grade at a later time. There will be no change in your final grade once it is submitted. Make up exams: No make up exams will be given in this course. You will obtain an average from all other exams, in case you fail to take a test due to some unexpected medical reasons or other. Please remember that satisfactory documentations must be provided in order to obtain an average, and if the documentation is not satisfactory, you will receive no points for that exam.