Genome 371 Lecture 11 - 1/26/11 4:17 PM Genome 371 Lecture...

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Unformatted text preview: 1/26/11 4:17 PM Genome 371 Lecture 11 Page 1 of 13 http://courses.washington.edu/au371mkr/resources/lecture_notes/lecture_11.html Lecture 11 DNA sequencing; Linkage 8 Nov 2010 Lecture 11 handout (pdf) Subscribe to podcast: Slide 1 Slide 2 Home Course mechanics Help hours Calendar Syllabus Lectures, Podcasts Quiz Sections Practice problems Exams GoPost Send email to class Useful links The Gradiator 1/26/11 4:17 PM Genome 371 Lecture 11 Page 2 of 13 http://courses.washington.edu/au371mkr/resources/lecture_notes/lecture_11.html Slide 3 Slide 4 Slide 5 Click to play animation (will open in a new window; may take a little while to load) From: "Genetics: From Genes to Genomes" by Hartwell et al. Slide 6 1/26/11 4:17 PM Genome 371 Lecture 11 Page 3 of 13 http://courses.washington.edu/au371mkr/resources/lecture_notes/lecture_11.html Slide 7 Slide 8 Slide 9 Slide 10 1/26/11 4:17 PM Genome 371 Lecture 11 Page 4 of 13 http://courses.washington.edu/au371mkr/resources/lecture_notes/lecture_11.html Slide 11 The sequence that is read is the sequence of the new strand as it is synthesized... complementary to the template strand that was used. Click here for a virtual tour (PowerPoint presentation) of the sequencing production line at the UW Genome Center in Fluke Hall. Slide 12 Slide 13 Since the DNA library was made with a particular plasmid vector, all clones in the library have the same vector "backbone" (highlighted in cyan in the picture on the left). We can therefore use the same sequencing primers to begin sequencing any clone we pull out of the library. The primers have to be chosen so that they will anneal (hybridize) in the vector portion but right next to the vector-insert junction; also, they have to be chosen to anneal to the correct strand so that the 3'OH of the primer will prime synthesis in the direction of the insert and not back into the vector. Note that the diagram on the right just illustrates how (1) sequence information derived from one sequencing reaction can be used to design a primer for another sequencing reaction, so as to sequence further into the insert; and (2) that the insert can be sequenced from both ends. As discussed in class, any one sequencing reaction would have only ONE primer (but millions of copies of that one primer, of course). 1/26/11 4:17 PM Genome 371 Lecture 11 Page 5 of 13 http://courses.washington.edu/au371mkr/resources/lecture_notes/lecture_11.html Slide 14 The sequence trace becomes ambiguous if more than one primer is present in one sequencing reaction (compare the top trace made with one primer vs. the bottom trace)... so a sequencing reaction can only contain one primer (i.e., a primer that specifically recognizes and binds to one specific location on the template)....
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Genome 371 Lecture 11 - 1/26/11 4:17 PM Genome 371 Lecture...

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