Chapter 9 – DNA-Based Information Technologies- Genomics – the study of genes on the scale of whole cells and organisms- proteomics – the study of proteins on the scale of whole cells and organisms9.1 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 anincrease 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 endonucleaseso Selecting a small molecule of DNA capable of self-replication (cloning vector) Typically plasmids or viral DNAso Joining two DNA fragments covalently by DNA ligase Recombinant DNAs – composite DNA molecules comprising covalently linked segments from two or more sourceso Moving recombinant DNA from the test tube to a host cello Selecting or identifying host cells that contain recombinant DNA- Recombinant DNA technology (genetic engineering) – the methods used to accomplish the above and related tasksRestriction 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 moleculeo Restriction endonucleases – recognize and cleave DNA at specific sequences to generate a set of smaller fragmentso DNA ligases – join the DNA fragment to be cloned to a suitable cloning vector to link the DNA molecule together- 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- Restriction-modification system – the restriction endonuclease and the corresponding methylase- Three types of restriction endonucleases:o I – large, multisubunit complex containing both the endonuclease and methylase activities Cleave DNA at random siteso Type II restriction endonucleases Require no ATP Cleave the DNA within the recognition sequence itselfo III – large, multisubunit complex containing both the endonuclease and methylase activities Cleave the DNA about 25 bp from the recognition sequence- Sticky ends – the unpaired strands resulting from staggered cuts made by restriction endonucleaseso 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 DNAo 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 endonucleaseo Catalyzes formation of new phosphodiester bonds using ATPo Base-pairing of sticky ends facilitates this reaction- Linkers – synthetic DNA fragments that can be inserted between the ends that are being ligated to create new DNA sequences- Polylinkers – inserted DNA fragments with multiple recognition sequences for restriction endonucleasesCloning Vectors Allow Amplification of Inserted DNA Segments- Three popular cloning vectors:o Plasmidso Bacteriophageso Bacterial artificial chromosomes- Plasmidso Circular DNA molecules that replicate separately from the host chromosomeo Transformation – a process by which plasmids can be introduced into bacterial cells 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 43oCo Electroporation – when cells are incubated with the plasmid DNA are subjected to a high-voltage pulse Transiently renders the bacterial membrane permeable to large moleculeso Use of selective markers to select cells that actually take up the plasmid DNAo pBR322 is an E. coli plasmid that is a useful cloning vector 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 placeswhere the plasmid can later be cut to insert foreign DNA- Small size, which facilitates entry into cells and manipulation of the DNAo Transformation becomes less successful as plasmid size increases- Bacteriophageso Bacteriophage is a good vector to clone somewhat larger DNA 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 itis between 40,000 and 53,000 bp longo Ensures packing of recombinant DNA onlyo In vitro packaging – 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 DNAo 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 specieso Yeast artificial chromosomes (YACS) – contain all the elements needed to maintain a eukaryotic chromosomes n the yeast nucleus A yeast origin of replication Two selectable markers Specialized sequences needed for stability and proper segregation of the chromosomes at cell divisiono 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 and ligated This mixture is used to transform treated yeast cells with very large DNA moleculeso YACs that lack a telomere at either end are rapidly degradedSpecific DNA Sequences Are Detectable by Hybridization- Probe – a labeled DNA or RNA fragment complementary to the DNA being soughtExpression of Cloned Genes Produces Large Quantities of Protein- Expression vectors – cloning vectors with the
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