Chapter_20 - Chapter 20 Biotechnology Overview The DNA...

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Chapter 20 Biotechnology
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Overview: The DNA Toolbox In recombinant DNA , nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule Methods for making recombinant DNA are central to genetic engineering , the direct manipulation of genes for practical purposes DNA technology has revolutionized biotechnology , the manipulation of organisms or their genetic components to make useful products 2
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Fig. 20-2 DNA of chromosome Cell containing gene of interest Gene inserted into plasmid Plasmid put into bacterial cell Recombinant DNA (plasmid) Recombinant bacterium Bacterial chromosome Bacterium Gene of interest Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Plasmid Gene of Interest Protein expressed by gene of interest Basic research and various applications Copies of gene Protein harvested Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth 2 4 1 3 A preview of gene cloning and some uses of cloned genes 3
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Fig. 20-3 Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones. 5 3 3 5 1 One possible combination Recombinant DNA molecule DNA ligase seals strands. 3 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. 2 Restriction enzyme and DNA ligase 4
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Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids RESULTS Colony carrying non- recombinant plasmid with intact lacZ gene One of many bacterial clones Colony carrying recombinant plasmid with disrupted lacZ gene Cloning Eukaryotic genes in bacterial plasmids 5 Fig. 20-4
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Fig. 20-5a Bacterial clones Recombinant plasmids Recombinant phage DNA or Foreign genome cut up with restriction enzyme (a) Plasmid library (b) Phage library Phage clones Genomic libraries 6
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cDNA library A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell A cDNA library represents only part of the genome— only the subset of genes transcribed into mRNA in the original cells 7 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail DNA strand Primer mRNA Degraded mRNA DNA polymerase cDNA
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BAC clones A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert BACs are another type of vector used in DNA library construction 8 (c) A library of bacterial artificial chromosome (BAC) clones Large plasmid Large insert with many genes BAC clone
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C 3 5 C C G G G A A A T T T Screening a Library for Clones Carrying a Gene of Interest
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This note was uploaded on 12/27/2011 for the course LSCI 103 taught by Professor K.y.liao during the Spring '11 term at National Cheng Kung University.

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Chapter_20 - Chapter 20 Biotechnology Overview The DNA...

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