LEC17 - 1 Announcements • Quiz this weekend after 5 pm...

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Unformatted text preview: 11/7/2008 1 Announcements • Quiz this weekend: after 5 pm today • Regrades will be finished this weekend – Pick up in core office Monday (LS2305) Sanger or Dideoxy Sequencing 1. DNA to be sequenced is first denatured 2. Oligonucleotide of known sequence is 2. Oligonucleotide of known sequence is hybridized to template DNA DNA to be sequenced 3’ 5’ 5’ 3’ Oligonucleotide as primer 5’ 3’ 11/7/2008 2 3. Template-primer is distributed in four different tubes 4. To each tube are added all the substrates for DNA synthesis plus one dideoxynucleoside triphosphate To all tubes: DNA template Primer dNTPs DNA polymerase 11/7/2008 3 5. In vitro DNA synthesis produces a collection of fragments, each terminating with the dideoxynucleoside (in this case ddG) 6. Fragments are denatured and separated on polyacrylamide gel. Bands are visualized by autoradiography 3’ 3 11/7/2008 4 Autoradiogram of Dideoxy Sequencing Gel 11/7/2008 5 Automated sequencing uses fluorescent labels instead of radioactive labels. ddA-green tag ddG black tag ddG-black tag ddT-red tag ddC-blue tag 1. One reaction containing all four dNTPs and all four labeled ddNTPs. 2. PCR used to amplify sequencing products (only 1 primer) 3. Fragments separated by electrophoresis in a single lane. 4. A fluorescent detector records the color of the passing bands and generates a sequence. 11/7/2008 6 Genomic and cDNA libraries • Today, most often the entire library is sequenced – Gives sequence of entire genome – Comparison of genomic DNA and cDNA assists in annotation of the genome (determining genes, promoters, etc) • One example of identifying specific gene from an organism that has not been sequenced yet Protein Æ gene....
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LEC17 - 1 Announcements • Quiz this weekend after 5 pm...

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