BE115+-+Lecture09 - Lecture 9 DNA Electrophoresis; Projects...

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Lecture 9 DNA Electrophoresis; Projects "Medical scientists are nice people, but you should not let them treat you." - August Bier 1
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RT-PCR PCR can be: Non-specific – random primers amplify all DNA present Specific – carefully chosen primers amplify only a portion of cDNA corresponding to the mRNA of the protein we’re interested in RT-PCR (as done in class) is interested in the expression of a single gene. 2
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3 mRNA (Collagen II) mRNA (GAPDh) mRNA (EGF receptor) mRNA (TGFalpha) Purified RNA RT (non-specific) cDNA cDNA (Collagen II) cDNA (GAPDh) cDNA (EGF receptor) cDNA (TGFalpha) PCR (specific) cDNA (Collagen II) cDNA (GAPDh) cDNA (EGF receptor) cDNA (TGFalpha) amplified cDNA (sense and antisense) corresponding to the part of the Collagen II cDNA between the primers
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DNA Electrophoresis Analogous to SDS-PAGE for nucleic acids instead of proteins Agarose gel instead of acrylimide Followed by: Staining of total DNA with a DNA-binding fluorescent dye like ethidium bromide or SYBR-safe Fluoresces strongly when bound to DNA/RNA Fluoresces weakly otherwise Blotting, then staining of specific DNA with oligonucleotide probes 4
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DNA Electrophoresis Run your RT-PCR samples through an agarose gel The gel also contains SYBR- safe, so the dye binding occurs during the electrophoresis We’re going to look for a strong band of those amplified fragments! 5
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Identification No RT Faint band <100 bp Sample Faint band ~700 bp Strong band ~500 bp Faint band <100 bp Control Band ~800 bp Strong band ~500 bp 6 no RT Sample Contr ol Ladder 2072 base pairs 1500 base pairs 600 base pairs 100 base pairs primers primers product(?) control cDNA amplified portion of cDNA (523 bp) non-specific amplification
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Interpretation of Gels What does no band in the Control lane mean? What does a band in the no RT lane mean? What does a band of unexpected size in the Sample lane mean? 7 no RT Sample Contr ol Ladder 2072 base pairs 1500 base pairs 600 base pairs 100 base pairs
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Why is this important? Students do PCR looking for a promoter gene indicative of bioengineered corn They find it in bioengineered and in a small % of the kernels of non- bioengineered corn! With results from inverse PCR, they claim that the transgenes in the bioengineered corn are hopping promiscuously around in the genome and inserting themselves willy-nilly 8
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Inverse PCR Start with genomic DNA Add a restriction enzyme Ligate the fragments 9 Gene of interest
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Perform PCR with primers pointed in the opposite direction Result: amplification of DNA that flanks the gene of interest Gives you an idea of where the gene of interest is! 10
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This note was uploaded on 10/14/2011 for the course BIOE 115 taught by Professor Haley during the Spring '11 term at City College of San Francisco.

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BE115+-+Lecture09 - Lecture 9 DNA Electrophoresis; Projects...

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