Misunderstandings About GenesBENG 500BENG 500--1111CSCE 510CSCE 510--33INTRODUCTION TO COMPUTATIONAL BIOLOGYINTRODUCTION TO COMPUTATIONAL BIOLOGYMolecular Biology ToolsMolecular Biology ToolsJin-Woo KimDepartment of Biological and Agricultural EngineeringThe University of Arkansas – FayettevilleMolecular Biology ToolsMolecular Biology ToolsA set of roughly six different laboratory techniques defines theA set of roughly six different laboratory techniques defines theentire discipline of molecular biologyentire discipline of molecular biology••Restriction Enzyme DigestsRestriction Enzyme Digests••Gel ElectrophoresisGel Electrophoresis••BlottingBlotting••CloningCloning••DNA SequencingDNA Sequencing••Polymerase Polymerase Chain ReactionChain ReactionRESTRICTION ENZYME DIGESTSRESTRICTION ENZYME DIGESTS• Restriction enzymes make sequence-specific cuts in DNA.Blunt Ends vs. Sticky Ends• Restriction enzymes allow researchers to manipulate DNA molecules in a specific fashion.• Restriction fragments lead to powerful new methods for sequencing DNA.Other EnzymesOther Enzymes••DNA DNA LigasesLigases••Reverse Reverse TranscriptasesTranscriptasesGEL ELECTROPHORESISGEL ELECTROPHORESIS• Separating DNA fragments according to electric charges and/or molecular sizes.o Agarose gel electrophoresis = Mainly according to electric chargeso Polyacrylamide gel electrophoresis = Mainly according to molecular sizesBLOTTING and HYBRIDIZATIONBLOTTING and HYBRIDIZATION• Telling which DNA fragment in a mixture contains a sequence of interest.Blotting TechniquesBlotting Techniques••Southern Blotting Southern Blotting ––DNADNA••Northern Blotting Northern Blotting ––RNARNA••Western Blotting Western Blotting ––ProteinsProteinsMICROARRAYMICROARRAY––A variant of the blotting techniquesA variant of the blotting techniquesCLONINGCLONING• Insertion of a piece of DNA into a cell in such a way that the DNA will be replicated and maintained• To clone a gene, the DNA must be placed into a carrier DNA molecule (vector) whose structure is immune to the degrading enzymes in cells.Genetic LibraryGenetic Library--A collection of genes, each of which is cloned into a vectorA collection of genes, each of which is cloned into a vectorGenomicDNA LibrarycDNALibraryDNA SEQUENCINGDNA SEQUENCING••To determine the order or sequence of the To determine the order or sequence of the component nucleotides of DNA fragments component nucleotides of DNA fragments (<1000 b)(<1000 b)••Two MethodsTwo MethodsooMaxamMaxam--Gilbert MethodGilbert Method––Chemical Chemical degradation to generate DNA degradation to generate DNA subfragmentssubfragmentsooSanger MethodSanger Method(Chain(Chain--Termination Method)Termination Method)--Incorporation of modified nucleotides that prevent a DNA Incorporation of modified nucleotides that prevent a DNA polymerasepolymeraseactivityactivityPOLYMERASE CHAIN REACTION (PCR)POLYMERASE CHAIN REACTION (PCR)••Making multiple copies of a specific segment of DNA by primers aMaking multiple copies of a specific segment of DNA by primers and DNA nd DNA polymerasepolymeraseMisunderstandings About GenesMisunderstandings About Genes• “Genes make proteins”Genes contain information for making proteins. However, proteins are made by other proteins that use the gene as a manufacturing guide. Proteins cannot manufacture proteins without guide, but the guide alone is not sufficient for protein production.• “Genes are on chromosomes”Genes are not “on” chromosomes. A chromosome is composed of a very long molecule of DNA that isbound to proteins named histones. A gene is a given length of DNA that is translated into a chain of amino acids. The genes plus the bound proteins are the chromosome.• “Genes replicate themselves”Genes do not replicate themselves. Proteins replicate genes using the gene as a guide.• “DNA is like a blue print”DNA is much more than architectural blueprints. DNA is like a blueprint because it contains information for building proteins just as architectural blueprints contain information for building a structure. However, DNA not only has information for building the structure of an organism but it also contains information for making enzymes, hormones, receptor molecules, transport proteins, and antibodies.• “Genes determine who we are”Genes do not determine who we are. Genes ‘influence’ who we are. Genes are only one factor that contribute to a trait (phenotype), many genes influence a single trait, and any one gene affects many