2010 Bio 320 Lecture 18 - 20

2010 Bio 320 Lecture 18 - 20 - "An isolated DNA coding for...

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“An isolated DNA coding for a BRCA1 polypeptide, said polypeptide having the amino acid sequence set forth in SEQ ID NO:2.” (Claim 1 of U.S. Patent No. 5,747,282). “Product of nature” a patentable chemical? or On March 29, 2010 a New York federal court ruled that the patents on the BRCA1 and BRCA2 genes are invalid .
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lecture 17 18 Genetic technology I: gene cloning why clone a gene? In order to do gene chemistry,  e.g ..... • DNA sequencing • probe labelling   & hybridization (where/when is it transcribed?) ...you need to isolate large amounts of a  specific  DNA segment. (where is the protein located?) • gene modification (what does the gene encode?) to find out:
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Genomes are  complex : up to 10 9  bp of non-repeating (unique) sequence. - any single gene (10 - 10 4 bp) is a  small proportion  of a genome….  The problem... The solution... …replicate  and purify smaller fragments of the genome.   …distinguished from the rest  only by its base  sequence Isolate..
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Two ways to replicate specific DNA sequences: 1 . Molecular cloning Cut genome into  fragments  Select the clone  of interest 2. Polymerase chain reaction Replicate only the specific fragment of interest Replicate all fragments as clones
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Techniques & tools Cut and paste:   join specific DNA segments from different sources Label:  add a traceable isotope/molecule to a DNA segment  Replicate :  make many identical copies of a DNA or RNA segment Hybridize : identify DNA/RNA segments by complementary base-pairing The toolbox: Restriction enzymes DNA ligase DNA polymerases Vectors Primers Probes
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Restriction enzymes - a bacterial defence against foreign DNA  Discovery: from the  restricted  host range of some phage.  E. coli strains infected with  bacteriophage  λ % successful infections: ~100%  K12  ~0.01%  Reinfect K12 with rare survivors: restriction ~100% !  modification
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Restriction and modification A methylase  modifies sites in bacterial DNA or surviving phage DNA,  - prevents cutting by endonucleases  • Enzyme names from bacterial species   •  Eco R1 E scherichia  co li, strain  R , enzyme  1    •  BamH1 B acillus  am yloliquefaciens...  An endonuclease  cuts invading bacteriophage DNA at specific sites.  • Restriction • Modification are due to pairs of enzymes that act on specific sites in DNA: 
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Isolating specific DNA fragments Restriction endonucleases cut DNA at specific base sequences NNNNNNNNNGGATCCNNNNNNNNN NNNNNNNNNCCTAGGNNNNNNNNN BamH1 5’ 5’ 3’ 3’ NNNNNNNNG NNNNNNNNCCTAG GATCCNNNNNNNN GNNNNNNNN 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ P O G P A 5 ' P O H 3 ' G Mo s t s ite s  have   dyadic  s ym m e try : re ad the  s am e  5’ to  3’ o n e ac h s tr
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Restriction sites BamH1 G/GATCC NNNNNNNNG NNNNNNNNCCTAG GATCCNNNNNNNN GNNNNNNNN 5’ 5’
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