320-10-18to20_1 - An isolated DNA coding for a BRCA1...

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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 (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 3 - 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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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 C % 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 Most sites have dyadic symmetry : read the same 5’ to 3’ on each str
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Restriction sites BamH1 G/GATCC NNNNNNNNG NNNNNNNNCCTAG GATCCNNNNNNNN GNNNNNNNN 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ NNNNNNNNCTGCA NNNNNNNNG GNNNNNNNN ACTGCNNNNNNNN 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ Pst1 CTGCA/G NNNNNNNNCCC NNNNNNNNGGG GGGNNNNNNNN CCCNNNNNNNN 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ Sma1 CCC/GGG Sau3A /GATC NNNNNNNNN NNNNNNNNNCTAG GATCNNNNNNNNN
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320-10-18to20_1 - An isolated DNA coding for a BRCA1...

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