MolCellEng_Notes6

MolCellEng_Notes6 - 1 Sanger sequencing by mass...

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1. Sanger sequencing by mass spectrometery 1. Slides 61 – 64 2. Chart of mass difference. Determine the template sequence using below mass spectrum and the mass table. Primer mass was 5000 Da. Mass Difference Last Base Incorporation A C G T A 312 286 328 373 C 311 285 327 372 1
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G 312 286 328 373 T 242 216 258 303 Pri mer 688 665 704 753 2
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Ans: CAGG Primer C A G G 5000 + 665 + 311 + 328 + 328 5665 5976 6304 6632 Find # in chart, note last base, record next base from top of chart. Use primer to start with and start adding to get next spectrum value. 3. Problem with linker arms with same length 1. Problem- fragments that end in T and C, peaks can be separated by as little as 1 Dalton 2. Solution – use a longer linker arm for ddTTP [Bio-16 linker to discriminate between C and T) 4. Capillary electrophoresis sequencing data has ambiguous peaks after the mutation site; BUT mass spectrometry results with clear peaks and accurate sequencing data before and after the mutation site. 2. New techniques for mapping and sequencing 1. Optical Mapping 1. Single molecule technique 2. Individual DNA molecules attached to glass support 3. Restriction enzymes on glass are activated 4. When DNA is cut, microscope records length of resulting fragments 3
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5. Has potential to rapidly generate restriction maps 2. Pyrosequencing http://www.youtube.com/watch?v=kYAGFrbGl6E 1. Based on production of pyrophosphate during sequencing reaction [DNA + dnTP, polymerase action, yields DNA plus one more nucleotide and PPi as byproduct] 2. Each time polymerase adds nucleotide (dNTP) to the growing strand, pyrophosphate (PPi) is released. 3.
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MolCellEng_Notes6 - 1 Sanger sequencing by mass...

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