Chapter 9 DNA Based Information Technologies Genomics proteomics the study of genes on the scale of whole cells and organisms the study of proteins on the scale of whole cells and organisms 9 1 DNA Cloning The Basics DNA cloning separating a specific gene or DNA segment from a larger chromosome attaching it to a small molecule of carrier DNA and then replicating this modified DNA thousands or millions of times through both an increase in host cell number and the creation of multiple copies of the cloned DNA in each cell Cloning entails five procedures o Cutting DNA at precise locations by sequence specific endonucleases o Selecting a small molecule of DNA capable of self replication cloning vector Typically plasmids or viral DNAs o Joining two DNA fragments covalently by DNA ligase Recombinant DNAs covalently linked segments from two or more sources composite DNA molecules comprising o Moving recombinant DNA from the test tube to a host cell o Selecting or identifying host cells that contain recombinant DNA Recombinant DNA technology accomplish the above and related tasks genetic engineering the methods used to Restriction Endonucleases and DNA Ligase Yield Recombinant DNA Two classes of enzymes lie at the heart of the classic approach to generating and propagating a recombinant DNA molecule o Restriction endonucleases sequences to generate a set of smaller fragments vector to link the DNA molecule together o DNA ligases recognize and cleave DNA at specific join the DNA fragment to be cloned to a suitable cloning The sequence of DNA that would be recognized by its own restriction endonuclease is protected from digestion by methylation of the DNA catalyzed by a specific DNA methylase corresponding methylase Restriction modification system the restriction endonuclease and the Three types of restriction endonucleases o I large multisubunit complex containing both the endonuclease and methylase activities Cleave DNA at random sites o Type II restriction endonucleases Require no ATP Cleave the DNA within the recognition sequence itself o III large multisubunit complex containing both the endonuclease and methylase activities Cleave the DNA about 25 bp from the recognition sequence Sticky ends restriction endonucleases the unpaired strands resulting from staggered cuts made by o Can base pair with each other or with complementary sticky ends of other DNA fragments Blunt ends created when restriction endonucleases cleave both strands of DNA at the opposing phosphodiester bonds leaving no unpaired bases at the ends The average size of the DNA fragment produced by cleaving genomic DNA with a restriction endonuclease depends on the frequency with which a particular restriction site occurs in the DNA o This depends largely on the size of the recognition sequence DNA ligase attaches the DNA fragment to a vector that was digested by the same restriction endonuclease o Catalyzes formation of new phosphodiester bonds using ATP o Base pairing of sticky ends facilitates this reaction synthetic DNA fragments that can be inserted between the ends Linkers that are being ligated to create new DNA sequences Polylinkers for restriction endonucleases inserted DNA fragments with multiple recognition sequences Cloning Vectors Allow Amplification of Inserted DNA Segments Three popular cloning vectors o Plasmids o Bacteriophages o Bacterial artificial chromosomes Plasmids o Circular DNA molecules that replicate separately from the host chromosome o Transformation bacterial cells a process by which plasmids can be introduced into The cells and plasmid DNA are incubated together at 0oC in CaCl2 solution then subjected to a shock by rapidly shifting the temperature to 37 to 43oC when cells are incubated with the plasmid DNA are o Electroporation subjected to a high voltage pulse Transiently renders the bacterial membrane permeable to o Use of selective markers to select cells that actually take up the o pBR322 is an E coli plasmid that is a useful cloning vector large molecules plasmid DNA Features An origin of replication ori required to propagate the plasmid and maintain it at a level of 10 to 20 copies per cell Two genes that confer resistance to different antibiotics Several unique recognition sequences that are targets for different restriction endonucleases providing places where the plasmid can later be cut to insert foreign DNA Small size which facilitates entry into cells and manipulation of the DNA o Transformation becomes less successful as plasmid size increases o Bacteriophage is a good vector to clone somewhat larger DNA Bacteriophages segments Features About one third of the genome is nonessential and can be replaced with foreign DNA DNA is packaged into infectious phage particles only if it is between 40 000 and 53 000 bp long o In vitro packaging o Ensures packing of recombinant DNA only packaging of recombinant DNAs into phage particles by adding them to crude bacterial cell extracts that contain all the necessary proteins Bacterial Artificial Chromosomes BACs o Plasmids designed for the cloning of very long segments of DNA o Generally include selective markers and a very stable origin of replication Yeast Artificial Chromosomes YACs o Shuttle vectors plasmids that can be propagated in cells of two or more different species needed to maintain a eukaryotic chromosomes n the yeast nucleus YACS contain all the elements o Yeast artificial chromosomes A yeast origin of replication Two selectable markers Specialized sequences needed for stability and proper segregation of the chromosomes at cell division o The genomic DNA is prepared by partial digestion with restriction endonucleases Fragments are then separated by pulsed field gel electrophoresis a variation of gel electrophoresis that allows the separation of very large DNA segments The DNA fragments are mixed with the prepared vector arms This mixture is used to transform treated yeast cells with very and ligated large DNA molecules o YACs that lack a telomere at either end are rapidly degraded Specific DNA Sequences Are Detectable by Hybridization a labeled DNA or RNA fragment complementary to the DNA being Probe sought Expression of Cloned Genes Produces Large Quantities of Protein Expression vectors signals needed for the regulated expression of a cloned gene cloning vectors with the transcription and translation The rate of expression of the cloned gene is controlled by replacing the gene s own promoter and regulatory sequences with more
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