Biotechnology Chapter 20 Flashcards

-by 2007 researchers had sequenced hundreds of prokaryotic genomes and human genome -key accomplishment in DNA technology-helped researchers sequence-DNA molecules formed when segments of DNA from two different sources--often different species--are combined in vitro (test tube)

-manipulation of organisms or their components to make useful products-long history that includes early practices of selective breeding of farm animals and using microorganisms to make wine and cheese-also encompasses genetic engineering

-direct manipulation of genes for practical purposes-launched revolution in biotechnology, expanding scope of potential applications -use of microarray analysis has allowed researchers to compare gene expression in different samples, such as those from cancerous and normal tissues

-methods for preparing well-defined segments of DNA in multiple identical copies-enables scientists to work directly with specific genes

-Bacteria have these small circular DNA molecules that replicate separately from the bacterial chromosome-has only a small number of genes that may be useful when bacterium is in particular environment but may not be required for survival under most conditions -used in cloning

-first isolate plasmid from bacterial cell and insert DNA from another source (foreign) into it-resulting plasmid is recombinant DNA -plasmid returned to bacterial cell-->recombinant bacterium-bacterium reproduces through repeated cell divisions to form clone of cells (foreign DNA cloned at same time)

-production of multiple copies of a single gene -useful to make copies of particular gene and to produce a protein product -can isolate copies for research or endow an organism with new metabolic capability (pest resistance) -most protein coding genes exist in only one copy per haploid genome--ability to prepare rare DNA fragments is crucial

-gene cloning and genetic engineering rely on these to to cut DNA molecules at a limited number of specific locations-restriction endonucleases -discovered in 1960s by studying bacteria -protect bacterial cell by cutting up foreign DNA-hundreds of different ones have been identified and isolated -specific, recognize particular DNA sequence

-particular DNA sequence recognized by restriction enzyme -cutting both DNA strands at precise points within this site -DNA of bacterial cell protected from cell's own restriction enzymes by addition of methyl groups to A or C within sequences recognized by enzymes-most are symmetrical (same when read in 5'-->3' direction)-most 4-8 nucleotides -may make many cuts in DNA molecule

-most useful restriction enzymes cleave sugar-phosphate backbones in the two DNA strands in a staggered manner -resulting double stranded fragments have at least one of thees single-stranded ends-these short extensions can form hydrogen-bonded base pairs with complementary ones on any other DNA molecules cut with same restriction enzyme -temporary

-the temporary associations formed by sticky ends can be made permanent by this enzyme-catalyzes formation of covalent bonds that close up the sugar-phosphate backbones of DNA strands

-original plasmid -DNA molecule that can carry foreign DNA into host cell and replicate there -plasmids can be isolated, reintroduced into cells, and they multiply rapidly

1) isolate genomic DNA from cells; isolate plasmid from bacterial cellsplasmid has been engineering to carry ampR and lacZ--restriction site is within lacZ gene2) plasmid and chromosomal DNA cut with same restriction enzyme3) fragments are mixed, allowing base pairing of complementary sticky ends. Add DNA ligase, covalently bonding endssome recombinant DNA fragments will contain gene of interest, some won't 4) DNA mixture added to bacteria that have mutation in lacZ gene on their own chromosome, making unable to hydrolyze lactose or x-galsome cells acquire recombinant plasmid, some nonrecombinant, fragment of noncoding DNA, or nothing at all 5) only cells with plasmid will reproduce, bc only they have ampR gene (resistance to amp on medium)colonies without recombinant DNA have lacZ intact, and will produce B-galactosidase--will be blue no functional B-galactosidase produced in colonies containing recombinant plasmids--will be white

-shotgun approach: no single gene targeted for cloning; thousands of recombinant plasmids produced (restriction enzyme)

-complete set of plasmid containing cell clones, each carrying copies of a particular segment of initial genome -each plasmid clone is like a "book" containing specific info-certain bacteriophages have been used as cloning vectors to make this