Chapter 20 PPT Notes 1 Biotechnology manipulation of organisms or their components to make useful products 2 Applications of DNA technology affect everything from agriculture to criminal law to medical research 3 Nucleic acid hybridization the base pairing of one strand of nucleic acid to the complementary sequence on another strand 4 Genetic engineering direct manipulation of genes for practical purposes 5 DNA sequencing exploit the principle of complementary base pairing to determine a gene s complete nucleotide sequence a next generation sequencing techniques use single template strand that is immobilized and amplified to produce an enormous number of identical fragments 6 DNA cloning scientists prepare well defined DNA segments in multiple identical copies by this process 7 Plasmids small circular DNA molecules that replicate separately from the bacterial chromosome 8 Recombinant DNA molecule with DNA from two different sources 9 Gene cloning production of multiple copies of a single gene is a type of DNA cloning a Useful for amplifying genes to produce a protein product for research medical or other purposes 10 Cloning vector plasmid used to clone a foreign gene a Bacterial plasmids widely used as cloning vectors because they re readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly 11 Restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites 12 Restriction fragments the cuts a restriction enzyme makes 13 Sticky ends fragments produced by the most useful restriction enzymes that cut DNA in a staggered way 14 DNA ligase enzyme that seals the bonds between restriction fragments 15 Gel electrophoresis technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties a Negatively charged DNA move toward the positive electrode smaller fragments move faster and further than larger ones 16 Polymerase Chain Reaction PCR produces many copies of a specific target segment of DNA a Taq polymerase used because it is a heat stable DNA polymerase b 3 steps heating cooling and replication 17 Expression vector cloning vector that contains a highly active bacterial promoter 18 Eukaryotic DNA cloning and expression systems can avoid eukaryote bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes 19 Electroporation applying a brief electrical pulse to create temporary holes in plasma membranes method of introducing recombinant DNA into euk cells a Scientists can inject DNA into cells using microscopically thin needles once in the cell the DNA is incorporated into the cell s DNA by natural genetic recombination 20 mRNA can be detected by nucleic acid hybridization with complementary molecules a molecules of either DNA or RNA are nucleic acid probes 21 In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism 22 Reverse transcriptase polymerase chain reaction RT PCR useful for comparing amounts of specific mRNAs in several samples at the same timed a Reverse transcriptase is added to mRNA to make complementary DNA cDNA serves as a template for PCR amplification of the gene of interest b Products ran on a gel and mRNA is identified 23 DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions a By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets 24 In vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function a When mutated gene is returned to cell the normal gene s function might be determined by examining the mutant s phenotype 25 RNA Interference RNAi the silencing of gene expression 26 SNPs single nucleotide polymorphisms single nucleotide variants are among the most useful genetic markers a SNP variants found frequently associated with a certain inherited disorder let researchers know its likely location for disease causing gene b SNPs are rarely directly involved in the disease they re mostly in noncoding regions of the genome 27 Stem cell relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells 28 Totipotent cell cell that can generate a complete new organism 29 Nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell 30 Pluripotent embryonic stem ES cells are capable of differentiating into many different cell types a Ultimate aim of research with stem cells is to supply cells for the repair of damaged or diseased organs 31 Induced pluripotent stem cells iPS cells used as models for study of certain dieseases and potentially as replacement cells for patients can perform most of the functions ES cells can perform 32 Transgenic animals made by introducing genes from one species into the genome of another animal 33 STRs are variations in the number of repeats of specific DNA sequences a PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
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