BIOL 2960 Lecture 28: Recombinant DNA Technology

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Washington University in St. Louis
Biology And Biomedical Sciences
Biology And Biomedical Sciences BIOL 2960
Kunkel Barbara

Lecture 28 Saturday, April 22, 2017 4:09 PM RECOMBINANTDNA TECHNOLOGY • Recombinant DNA technologywas the precursor to all major biological technologies! ○ Isolate genes ○ Characterize genes ○ Genetic engineering ○ New technologies(PCR, DNA synthesis, etc.) ○ Genomics • OUTLINE: a. Molecular Cloning b. Recombinant DNA c. Genomic and cDNA libraries d. Cloning by PCR • MolecularCloning ○ A clone is a population of identical items that are derived from a single progenitor. Items can be: ▪ Genetically identical bacterial cells (a colony) ▪ Genetically identical individuals (e.g. Dolly the sheep) ▪ DNA molecules: "molecular cloning" technologyallows production of a large quantity of a particular DNA molecules □ Recombinant DNA Technology □ PolymeraseChain Reaction (PCR) • RecombinantDNA Technology ○ Step 1: Create chimeric recombinant DNA molecules in a test tube by a "cut and paste" mechanism (vector+ cargo DNA) ▪ Vector: DNA capable of autonomousreplication in a host cell □ A vectoris something that carries something from one host to another □ Requirements:  Capable of autonomous(not linked to host DNA) DNA replication after introduction into host cell  Capable of carrying foreign DNA = tolerates the insertion of extraneous DNA at one or more positions  Contains at least one genetic marker (selectableor screenable) to indicate the vector's presence in the host  Genomethat is well-characterized, of a limited size, and single nucleic acid molecule □ The most popular cloning vectorsare based on plasmids, but bacteriophage based vectors were used most in early studies  Plasmid: double-stranded circular DNA moleculesthat replicate independently of the bacterial chromosome  Examples: ◊ Resistance or R factors: plasmids conferring antibiotic resistance -- what we used in lab ◊ Fertility or F factors: ~100 kb plasmid in E. coli that is responsible for bacterial mating -- carries trait into the bacteria that it is mating with, so replicated through mating  Plasmid with a multiple copy origin of replication: plasmid replicates until it reaches about 300-500copies inside cell Multiple cloning site: cloning site is region where foreign DNA can be  Multiple cloning site: cloning site is region where foreign DNA can be inserted -- multiple implies that there are multiple places within the site that an enzyme can cut so different segments can be put in ◊ These only really work with inserts up to 10kb in size. Any bigger and the vector starts messing up. ◊ If you want to analyze a large piece of DNA, you should use a plasmid that has an origin that replicates at one copy per cell at a time -- allows stable maintenance with large fragments of human DNA (>100kb).  E.g. BAC (bacterial artificial chromosome)vector has an origin from F factor ▪ Cargo/insert: any DNA of interest from any source □ ○ Step 2: Isolation and amplification of recombinantmolecules in a host that supports replication of the vector ○ How it works: 1. Plasmid DNA is introduced into a cell via transformation:direct delivery of DNA into a cell 2. The plasmid replicates and after exponential growth, there is amplification of the plasmid yielding many cells (10^9/ml)with many plasmids (300-500per cell) ○ How are DNA molecules cut and pasted? ▪ DNA is cut using restriction endonucleases ▪ Restriction endonucleases/enzymesare proteins that recognize specific DNA sequences and cleave both sugar-phosphate backbones at specific positions relative to their recognition sequence ▪
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