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Chapter44 Recombinant DNA Technology Outline Introduction to recombinant DNA technology Detailed steps of genetic cloning Applications of genetic cloning PCR Mutagenesis and Protein engineering Analysis of DNA–protein interactions Analysis of DNA–protein interactions Analysis of gene function Expression of Proteins Using Recombinant DNA Technology and other uses SELEX Biochips Genomics overview Definition of recombinant DNA Production of a unique DNA molecule by joining together two or more DNA fragments not normally associated with each other DNA fragments are usually derived from different biological sources Steps of genetic cloning Isolation and fragmentation of the source DNA. The source DNA can be total genomic DNA from an organism of interest, DNA synthesized from an RNA template by reverse transcriptase, a gene or genes amplified by the polymerase chain reaction, or even wholly synthetic DNA made in vitro. If genomic DNA is the source, it is first cut with restriction enzymes to give a mixture of fragments of manageable size. These products of digestion are inserted into a DNA molecule called a vector ? Enables desired fragment to be replicated in cell culture to very high levels in a given cell (copy #) Introduction of recombinant DNA molecule into an appropriate host cell Transformation or transfection Each cell receiving rDNA = CLONE May have thousands of copies of rDNA molecules/cell after DNA replication As host cell divides, rDNA partitioned into daughter cells Population of cells of a given clone is expanded, and therefore so is the recombinant DNA Some enzymes used in recombinant DNA technology Restriction Endonucleases Origin and function Bacterial origin = enzymes that cleave foreign DNA Named after the organism from which they were derived EcoR I from Escherichia coli BamH I from Bacillus amyloliquefaciens Protect bacteria from bacteriophage infection Restricts viral replication Bacterium protects it’s own DNA by methylating those specific seque
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