Integrated Genomics: A Discovery-Based Laboratory Course / Edition 1

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Overview

Integrated Genomics: A Discovery-Based Laboratory Course introduces the excitement of discovery to the basic molecular biology laboratory. Utilizing up-to-date molecular biology protocols and a basic experimental design, this text offers experience with three different model systems. Students will become familiar with the simplicity and power of single-celled organisms, Escherichia coli and Saccharomyces cerevisiae, as they search for genes that interact and function within the nematode Caenorhabditis elegans.

Incorporated throughout the course are exercises designed to offer students familiarity with the wealth of bioinformatics data that can be accessed on the World Wide Web. Following completion of interaction studies within the yeast, the course is designed to allow students to examine the functional consequences of reducing a gene’s function within the multicellular worm that is both simple and inexpensive to maintain within a laboratory. The inclusion of alternative experiments allow for flexibility in determining the ending date or goal of the laboratory, as well as working within the available budget and resources of most any classroom environment.

Further striking features of this title are:

  • An accompanying Web site providing PowerPoint slides, plus links to the internet, and regular updates as bioinformatics databases evolve and methods improve. www.wiley.com/go/caldwell
  • Inclusion of modern genomic/proteomic technologies such as the yeast two-hybrid system and RNAi
  • Detailed experimental protocols and easy access to instructional materials

This discovery-based laboratory course provides excellent practical training for those pursuing career paths in biomedicine, pharmacy, and biotechnology.

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Editorial Reviews

From the Publisher
"I greatly admire the efforts of the authors. Their goals are praiseworthy and this manual is well written." (The Quarterly Review of Biology, September 2007)
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Product Details

  • ISBN-13: 9780470095010
  • Publisher: Wiley
  • Publication date: 9/25/2006
  • Edition number: 1
  • Pages: 246
  • Product dimensions: 8.80 (w) x 11.12 (h) x 0.81 (d)

Meet the Author

Guy A. Caldwell, PhD, is an Associate Professor in theDepartment of Biological Sciences at the University of Alabama,where since 1999 he has held an undergraduate professorialappointment from the Howard Hughes Medical Institute. He holds anadjunct appointment at the University of Alabama at Birmingham, asan Assistant research Professor of Neurology. In 2001, Dr Caldwellwas named a Bail O' Connor Scholar of The March of Dimes BirthDefects Foundation for his research into the molecular basis ofchildhood birth defects of the brain. Dr Caldwell is a recipient ofgrants from The March of Dimes, National Institutes of Health,Dystonia Medical Research Foundation, Parkinson's DiseaseFoundation, National Parkinson Foundation, and the Bachmann-StraussDystonia & Parkinson Foundation. In 2003, The CaldwellLaboratory was selected as 1 0f 11 groups worldwide to representthe research goals of the Michael J. Fox Foundation for Parkinson'sresearch in their Protein Degradation Grant Initiative. For hiscombines teaching and research efforts, Dr Caldwell was also chosenas the recipient of a 2003 CAREER Award from the National ScienceFoundation. In 2005, he was named Alabama Professor of the Year bythe Carnegie Foundation for the Advancement of Teaching and Councilfor Advancement and Support of Education. Dr Caldwell, a native ofthe New York City area, received his undergraduate degree inBiology from Washington & Lee University in 1986 and his PhD inCell, Molecular & Developmental Biology from The University ofTennessee in 1993. Following receipt of his doctorate, he moved toColumbia University in New York where he was twice named therecipient of fellowships from the National Institute ofNeurological Disease and Stroke. He is the author of two editionsof a widely adopted textbook, Biotechnology: A Laboratory Course,published worldwide in three languages. He teaches courses inIntegrated Genomics, Neuronal Signaling, General biology, and anacclaimed seminar on the societal impact of the Human GenomeProject.

Shelli N. Williams, PhD, is a research scientists at aprivate forensic company based in Virginia. Following her earlygraduation Magna cum laude from undergraduate studies, Dr Williamsbegan her graduate work in the laboratory of Drs Guy and KimCaldwell at The University of Alabama, where she earned herdoctorate from The University of Alabama in 2006. Dr Williamsserved as an adjunct faculty member in New College, aninterdisciplinary department at The University of Alabama, whereshe was the instructor of a seminar course demonstrating how thenature of the laboratory experience plays an essential role in theunderstanding and advancement of science. She has experience playsan an essential role in the understanding and advancement ofscience. She has experience teaching introductory biology coursesto both majors and non-majors students and has been a repeatedguest lecturer in a cross-disciplinary bioethics class. As a PhDcandidate, Dr Williams served as a teaching assistant forIntegrated genomics, a discovery-based genomics course funded bythe Howard Hughes Medical Institute. Dr Williams was named therecipient of two university-wide Graduate Council Fellowships, aswell as receiving recognition as an Isabella Hummel graham Scholarhonoring outstanding female students throughout the university. Shealso received a competitive Worthington Biochemical Travel Awardfrom the American Society of Cell Biology, Placing her among thehighest honored student researchers at their 2003 Conference.Subsequent graduate work establishing Caenorhabditis elegans as amodel for epilepsy was high-lighted in news releases by the HowardHughes Medical Institute. In recognition of her accomplishments, DrWilliams was awarded the 2005 Joab Langston Thomas Award, the tophonor for PhD students in Biological Sciences at the University ofAlabama.

Kim A. Caldwell, PhD, is an Assistant Professor in theDepartment of Biological Sciences at The University of Alabama. DrCaldwell is a Faculty Affiliate of The University of Alabama Centerfor Green Manufacturing and she is an Adjunct research AssistantProfessor in the Department of Neurology at the University ofAlabama at Birmingham Medical School. Dr Caldwell, a native of theBuffalo area, received her undergraduate degree in recombinant GeneTechnology from the State University of New York at Fredonia andher MS and PhD degrees in Biotechnology and Cell, Molecular &Developmental Biology, respectively, from The University ofTennessee, While at Tennessee, Dr Caldwell was a four-timerecipient of the Oak Ridge National Lab-UT Science Allianceteaching/research Award and the Chancellor's Award forExtraordinary Professional Promise. Following receipt of herdoctorate, she held postdoctoral research appointments at theRockefeller University and Columbia University in New York, duringwhich time he was named the recipient of a Revson Fellowship and aNational research Service Award from the National Institue of ChildHealth and Human Development. Her research has been published inmany outstanding peer-reviewed journals, including Nature,Proceedings of the National Academy of Sciences, Journal ofNeuroscience, Human Molecular Genetics the Journal of Cell Scienceand Development. Dr Caldwell serves as Director of the HowardHughes Medical Institute rural Science Scholars program at Alabama.Additionally, she has designed and taught courses in GeneralBiology, a seminar on the societal impact of the Human GenomeProject, and a course entitled ' the Language of research', whichshe teaches jointly for Howard Hughes research Interns at bothStillman College and the University of Alabama. For her teachingefforts,in 2005 Dr Caldwell was selected as a Education Fellow inthe Life Sciences of the National Academy of Sciences.

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Table of Contents

Preface.

Author biographies.

Acknowledgments.

List of figures.

1 Introduction to basic laboratory genetics.

1.1 Transferring and handling C. elegans.

1.2 Introduction to laboratory genetics.

2 Gene expression analysis using transgenic animals.

2.1 Transgenic gene expression analysis in C. elegans:lacZ staining.

2.2 Transgenic gene expression analysis in C. elegans:GFP analysis.

3 Creation and testing of transgenic yeast for use inprotein–protein interaction screening.

3.1 Small-scale transformation of S. cerevisiae.

3.2 Transformation of S. cerevisiae to test fornon-specific interaction.

3.3 Assaying for protein–protein interaction by reportergene expression.

4 Yeast two-hybrid screening.

4.1 Protein–protein interaction screening of a C.elegans cDNA library.

4.2 Assaying for protein–protein interaction by reportergene expression.

5 Isolation and identification of interactingproteins.

5.1 Preparation of electrocompetent E. coli.

5.2 Isolation of DNA from yeast and electroporation of E.coli.

5.3 Small-scale isolation of plasmid DNA from E. coli:the mini-prep.

5.4 Sequencing of two-hybrid library plasmid DNA vectors.

6 Using bioinformatics in modern science.

6.1 DNA sequence chromatogram.

6.2 BLASTing your sequence.

6.3 Evaluating sequence results and choosing an RNAi target.

6.4 Bioinformatics practice questions.

7 Generation of an RNAi vector.

7.1 Small-scale isolation of genomic DNA from C.elegans.

7.2 PCR amplification of target gene sequence from C.elegans genomic DNA.

7.3 Preparations for cloning to generate RNAi vector.

7.3.1 Agarose gel electrophoresis.

7.3.2 Removal of dNTPs from PCR reaction.

7.3.3 Restriction enzyme digestion of PCR product and C.elegans RNAi vector.

7.4 Gel purification of DNA and ligation of vector andPCR-amplified DNA.

7.4.1 Preparative agarose gel electrophoresis.

7.4.2 Gel purification of DNA from agarose gel.

7.4.3 Ligation of vector and PCR-amplified DNA.

7.5 Transformation of ligation reactions.

7.6 PCR screening of transformation colonies.

7.7 Small-scale isolation of plasmid DNA from E. coli:the mini-prep.

7.8 Verifying successful ligation by restriction digestion.

8 RNA-mediated interference by bacterial feeding.

8.1 Preparation of RNAi-feeding bacteria for transformation.

8.2 Media preparation for RNAi feeding.

8.3 Transformation of RNAi-feeding strain HT115(DE3).

8.4 RNA interference by bacterial feeding of C.elegans.

8.5 Analyzing effects of dsRNAi.

8.5.1 Assaying for sterility (Ste) or embryonic lethality(Emb).

8.5.2 Assaying for growth effect.

8.5.3 Assaying for morphological effects.

8.5.4 Assaying for general neuromuscular effects.

8.5.5 Assaying for specific neuronal effects.

8.5.6 Assaying for dauer formation.

Appendix I Recombinational cloning.

AI.1 Isolation of genomic DNA from C. elegans.

AI.2 PCR amplification of target gene sequence from C.elegans genomic DNA.

AI.3 Agarose gel electrophoresis and clean-up of PCRreaction.

AI.4 Entry vector cloning.

AI.5 Small-scale isolation of plasmid DNA from E. coli:the mini-prep.

AI.6 Destination vector cloning.

AI.7 Small-scale isolation of plasmid DNA from E. coli:the mini-prep.

Appendix II Recipes and media preparation.

Solution recipes.

Media preparation.

Appendix III Sterile techniques and worm protocols.

Sterile techniques.

Worm protocols.

Appendix IV Mutant C. elegans phenotypes.

Appendix V Vector maps.

Subject index.

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