Biotechnology and Recombinant DNA(Chapter 9)Lecture MaterialsforAmy Warenda Czura, Ph.D.Suffolk County Community CollegeEastern CampusPrimary Source for figures and content:Tortora, G.J. Microbiology An Introduction 8th, 9th, 10th ed. San Francisco: PearsonBenjamin Cummings, 2004, 2007, 2010.Amy Warenda Czura, Ph.D.1SCCC BIO244 Chapter 9 Lecture SlidesBiotechnology = use of microbes, cells, or cellcomponents to make a productGenetic engineering = inserting genes into cells, this involves:Recombinant DNA technology = the techniques for making recombinant DNARecombinant DNA (rDNA) = DNA assembled in the lab that contains the desired genes(e.g. insulin to express in bacteria fordisease therapy)(e.g. viral envelope genes for expressionin yeast for vaccine use)Amy Warenda Czura, Ph.D.2SCCC BIO244 Chapter 9 Lecture SlidesCloning = extracting or copying a gene ofinterest from its genomic source and putting it in an expression vector. Steps:1. Obtain the gene (PCR, restriction digest)2. Ligate it into a vector(vector = carrier piece of DNA)3. Transform the new recombinant DNA intobacteria/cells4. Grow up a population of transformed cellsthat contain the DNA (cells = clones)5. A. Harvest large quantities of the DNA foruse in other cells (cloning, gene therapy, etc), or more commonly:B. Harvest the gene product (protein) fromthe clones (therapeutics, vaccines)Amy Warenda Czura, Ph.D.3SCCC BIO244 Chapter 9 Lecture SlidesTools for Genetic Engineering1. Restriction enzymes-DNA cutting enzymes produced by bacteria-Cut DNA at specific base sequences-for cloning, use 4, 6, or 8 base cutters that leave “sticky ends”sticky ends = single stranded overhangs ofDNA that will base pair and re-ligate easilyAmy Warenda Czura, Ph.D.4SCCC BIO244 Chapter 9 Lecture Slides-hundreds of restriction enzymes are known-each cuts a unique sequence of DNA-easy to find one that cuts your target DNA and cloning vector2. Vectorsvectors = small pieces of DNA used forcloning-must be self replicating or integrate intothe genome-must be small enough to be manipulated in vitro-must have a selectable marker so transformedcells can be isolated (e.g. antibiotic resistance gene)-must have restriction enzyme cutting sites toclone genes into (e.g. a MCR: multiple cloning region)Amy Warenda Czura, Ph.D.5SCCC BIO244 Chapter 9 Lecture Slides-most vectors are plasmids (self replicating circles of DNA)Shuttle vector = plasmid capable of functioning in multiple species (needsmultiple selection markers, different promoters, etc)-some are based on viral DNA and are used tointegrate permanently into the genome ofthe transformed cells (e.g. Retroviruses, Adenoviruses, Herpesviruses)Amy Warenda Czura, Ph.D.6SCCC BIO244 Chapter 9 Lecture Slides3. Polymerase Chain Reaction (PCR)-used to amplify/copy DNA-can turn a few copies of DNA into billions ina few hours (exponential multiplication)DNA synthesis in a tube requires:1. template DNA (gene you want to copy)2. primers specific for the template3. free nucleotides (dNTPs)4. DNA polymeraseReplication process:1. Denature: reaction is heated to 94-95°Cto separate the ds DNA into ss templates2. Anneal: temperature is lowered to 50-60°C to allow primers to complementary base pair to the ss template DNA3. Extend: temperature is raised to 72°C toallow DNA polymerase to synthesize complementary strandsSteps repeated 20-30 timesAmy Warenda Czura, Ph.D.7SCCC BIO244 Chapter 9 Lecture Slides-each new DNA can serve as template in thenext round/cycle of replication:1 template x 30 rounds = 1.1x109 molecules-PCR only good for DNA ~4kb or less (a gene, not whole genome)Amy Warenda Czura, Ph.D.8SCCC BIO244 Chapter 9 Lecture Slides-PCR used for:1. cloning a gene out of an organism to express in a vector2. to detect low levels of an infectious agent3. to make more DNA from a limited sample for further analysisTechniques for Genetic Engineering1. Obtaining DNA of interestA. Prokaryotes-either cut out of genome with restriction enzymes or-PCR copies from the genomeB. Eukaryotes-get by reverse transcription:-eukaryotic genes have introns between thecoding exons-during transcription both introns and exons are copied into RNAAmy Warenda Czura, Ph.D.9SCCC BIO244 Chapter 9 Lecture Slides-exons are then spiced together to formmRNA for translation-prokaryotes cannot splice: eukaryoticgenes must have introns removed before expression in bacteria-yeast are eukaryotes and can splice, butoften splice human genes incorrectly,thus it is best to splice genes beforeexpression in yeast as wellAmy Warenda Czura, Ph.D.10SCCC BIO244 Chapter 9 Lecture SlidesReverse transcription:-reverse transcriptase (enzyme from retrovirus) is used to make a DNA copy from a spliced mRNA-the intron-free DNA is called cDNA-cDNA can be used for cloning in prokaryotesProcess: RT-PCR1. collected spliced mRNA from humancells2. Add:-primers complementary to the endsof the mRNA sequence to be copied-enzyme:reverse transcriptase: binds primersand synthesizes DNA copy-dNTPs3. Incubate 37°C: ssRNA → ssDNA4. Add DNA polymerase, run PCR cyclesto make many ds copies of the DNA= cDNAAmy Warenda Czura, Ph.D.11SCCC BIO244 Chapter 9 Lecture SlidesAmy Warenda Czura, Ph.D.12SCCC BIO244 Chapter 9 Lecture Slides2. Inserting foreign DNA into cells =Transformation-in order to be transformed a cell must first bemade competent to take up foreign DNA:A. Chemicals-CaCl2 makes pores in cell membraneB. Electroporation-electric shock forms temporary holes in membraneC. Gene gun-microscopic particlesof gold are coatedwith the DNA andshot with a burst ofhelium into cellshttp://upload.wikimedia.org/wikipedia/commons/7/70/Electroporation_Diagram.pngAmy Warenda Czura, Ph.D.13SCCC BIO244 Chapter 9 Lecture SlidesD. Microinjection-use tiny needle to inject DNA into thecell-the inserted DNA, regardless of method, mustbe on a self replicating plasmid or integrated into the host genome-if not it will be degraded and lost from the cell (no transformed cells, no clones)Amy Warenda Czura, Ph.D.14SCCC BIO244 Chapter 9 Lecture Slides3. Selecting a clone-few ligation events produce desired result (your gene in a vector)-transformation is very low frequency event-few cells will be transformed with desired vector-need to select those that 1)have vector 2)withyour geneBlue/White Screening:-plasmid contains AmpR so that all bacteria transformed with vector will survive on ampicillin (non-transformed cells die)-plasmid contains lacZ gene to codeβ-galactosidase-β-gal enzyme