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UMass Amherst MICROBIO 310 - Genetic Engineering

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Microbio 310 1st Edition Lecture 13 Outline of Last Lecture I. 10.6 Genetic RecombinationII. 10.7 TransformationIII. 10.8 TransductionIV. 10.9 Conjugation: Essential FeaturesV. 10.10 The Formation of Hfr Strains and Chromosome MobilizationVI. 10.13 Mobile DNA: Transposable ElementsOutline of Current Lecture I. 11.1 Restriction and Modification EnzymesII. 11.2 Nucleic Acid HybridizationIII. 11.3 Essentials of Molecular CloningIV. 11.4 Molecular Methods for MutagenesisV. 11.5 Gene Fusions and Reporter GenesVI. 11.6 Plasmids as Cloning VectorsCurrent Lecture11.1 Restriction and Modification Enzymes• Genetic engineering: using in vitro (outside of the body) techniques to alter genetic material inthe laboratory– Basic techniques include:These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.• Restriction enzymes• Gel electrophoresis• Nucleic acid hybridization• Nucleic acid probes• Molecular cloning• Cloning vectors• Restriction enzymes: recognize specific DNA sequences and cut DNA at those sites– Widespread among prokaryotes (original bacterial defense mechanism)– Rare in eukaryotes– Protect prokaryotes from hostile foreign DNA (e.g., viral genomes)– Essential for in vitro DNA manipulation• Three classes of restriction enzymes– Type II cleave DNA within their recognition sequence and are most useful for specific DNA manipulation• Restriction enzymes recognize inverted repeat sequences (palindromes)– Typically 4–8 base pairs long; EcoRI recognizes a 6- base-pair sequence• Sticky ends or blunt ends• Restriction enzymes protect cell from invasion by foreign DNA– Destroy foreign DNA– Must protect their own DNA from inadvertent destruction• Modification enzymes: protect cell’s DNA for restriction enzymes– Chemically modify nucleotides in restriction recognition sequence– Modification generally consists of methylation of DNA (adding methyl group to nucleotides)• Gel electrophoresis: separates DNA molecules based on size– Electrophoresis uses an electrical field to separate charged molecules– Gels are usually made of agarose, a polysaccharide– Nucleic acids migrate through gel toward the positive electrode due to their negatively charged phosphate groups– Small molecules move faster in gel than larger molecules so small pieces are at the bottom• Gels can be stained with ethidium bromide and DNA can be visualized under UV light• The same DNA that has been cut with different restriction enzymes will have different bandingpatterns on an agarose gel• Size of fragments can be determined by comparison to a standard- Restriction map: a map of the location of restriction enzyme cuts on a segment of DNA11.2 Nucleic Acid Hybridization• Nucleic acid hybridization: base pairing of single strands of DNA or RNA from two different sources to give a hybrid double helix– Segment of single-stranded DNA that is used in hybridization and has a predetermined identity is called a nucleic acid probe• Southern blot: a hybridization procedure where DNA is in the gel and probe is RNA or DNA– Northern blot: RNA is in the gel (will degrade if you are not careful)11.3 Essentials of Molecular Cloning• Molecular cloning: isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated• Three main steps of gene cloning: 1. Isolation and fragmentation of source DNA– Source DNA can be genomic DNA, RNA, or PCR-amplified fragments• Genomic DNA must first be restriction digested2. Insertion of DNA fragment into cloning vector – Most vectors are derived from plasmids or viruses– DNA is generally inserted in vitro– DNA ligase: enzyme that joins two DNA molecules• Works with sticky or blunt ends3. Introduction of cloned DNA into host organism– Transformation is often used to get recombinant DNA into host– Some cells will contain desired cloned gene, while other cells will have other cloned genes• Gene library: mixture of cells containing a variety of genes– Shotgun cloning: gene libraries made by cloning random genome fragments• Essential to detect the correct clone• Initial screen: antibiotic resistance, plaque formation– Often sufficient for cloning of PCR-generated DNA sequences• If working with a heterogeneous gene library you may need to look more closely• If cells express the foreign gene and its expression can be detected, then screening is relativelyeasy– Antibodies• Blood serum proteins produced by animals• Made by injecting animal with specific protein antigen• May need to look for the DNA if gene is not expressed– Nucleic acid probes• Look for binding of labeled nucleic acid probe to DNA from specific colonies11.4 Molecular Methods for Mutagenesis• Synthetic DNA– Systems are available for de novo synthesis of DNA (making synthetic DNA)– Oligonucleotides of 100 bases can be made– Multiple oligonucleotides can be ligated together– Synthesized DNA is used for primers and probes, and in site-directed mutagenesis• Conventional mutagens produce mutations at random• Site-directed mutagenesis: performed in vitro and introduces mutations at a precise location– Can be used to assess the activity of specific amino acids in a protein– Structural biologists have gained significant insight using this tool11.5 Gene Fusions and Reporter Genes• Reporter genes– Encode proteins that are easy to detect and assay• Examples: lacZ, luciferase, GFP genes• Gene fusions– Promoters or coding sequences of genes of interest can be swapped with those of reporter genes to make clear gene regulation under various conditions 11.6 Plasmids as Cloning Vectors• Plasmids are natural vectors and have useful properties as cloning vectors– Small size; easy to isolate DNA– Independent origin of replication– Multiple copy number; get multiple copies of cloned gene per cell– Presence of selectable markers (you can find them if they have a marker)• Vector transfer carried out by chemical transformation or electroporation• pUC19 is a common cloning vector– Modified ColE1 plasmid• Contains ampicillin resistance and lacZ genes• Contains polylinker (multiple cloning site) within lacZ gene• Blue/white screening– Blue colonies do not have vector with foreign DNA inserted– White colonies have foreign DNA inserted• Insertional inactivation: lacZ gene is


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UMass Amherst MICROBIO 310 - Genetic Engineering

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