1 Materials & Methods 5-1

1 Materials & Methods 5-1 - Molecular Biology...

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1 Molecular Biology PCR and DNA Markers
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2 Kary Mullis, after winning Nobel Prize for inventing PCR www.palamito.it/fotopalfiles
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3 PCR: Polymerase Chain Reaction “Amplify” DNA by in-vitro ( in plastico ) synthesis Key requirements: enzyme: Taq DNA polymerase, not denatured at high temps used to denature DNA primers: short (~ 20 b) oligonucleotides bind to denatured DNA, required to start DNA synthesis
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13 PCR amplifies the amount of DNA— doubles each cycle, so it increases exponentially
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14 Scientific American
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15 With the addition of thermostable Taq polymerase, PCR was adopted immediately, in many different areas of biology.
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18 PCR: polymerase chain reaction Advantages: powerful: works with miniscule amounts of DNA (even single cells) highly specific targets many primers work with broad range of targets (plants, animals, fungi) adapted to simplify DNA sequencing Disadvantages: difficulty / expense of primer development troubleshooting when reactions fail
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Thermosequencing: dideoxy sequencing + PCR Recall fundamental step of Sanger sequencing is enzymatic replication of DNA PCR using only one primer, plus different fluorescent nucleotides for each of 4
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1 Materials & Methods 5-1 - Molecular Biology...

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