CMSC423: Bioinformatic Algorithms, Databases and ToolsLecture 3Molecular biology primerWriting bioinformatics softwareCMSC 423 - Fall 2008 2Polymerase chain reaction (PCR)1. Denature2. Anneal (attachprimer)3. Extend4. RepeatCMSC 423 - Fall 2008 3How does PCR work?• 1. Start: 1 double-stranded molecule• 1. Denature: 2 single-stranded molecules• 1. Anneal: 2 single-stranded molecules with primers attached•1. Extend: 2 double-stranded molecules – one “long” (L) strand and one “short” (S) (terminated at a primer)• 2. Start: 2 double-stranded molecules: L+S, L+S• 2. Denature: 2 x L strands, 2 x S strands • 2. Anneal: all strands with primers attached• 2. Extend: 2 double-stranded molecules: L+S, L+S, 2 double-stranded molecules: S+SS, S+SSSS – strand terminated at both ends with a primerCMSC 423 - Fall 2008 4PCR Recurrences•Ln, Sn, SSn - # of strands of each type at cycle n•Ln = Ln – 1 = 2•Sn = Sn – 1 + Ln – 1 = Sn – 1 + 2 = 2 * (n – 1) = O(n)•SSn = Sn-1 + 2 * SSn – 1 = O(2n)•The sequence between the primers (SS) is amplified exponentially – will quickly overtake the solutionCMSC 423 - Fall 2008 5Quantitative PCR• Measure # of PCR cycles needed to reach a certain concentration of DNA – depends on initial # of molecules•Used in diagnostics: e.g. is this a random Anthrax spore from the environment or lots of spores from an attackhttp://www.dxsgenotyping.com/technology_main.htmCMSC 423 - Fall 2008 6DNA sequencing• Most techniques “trick” the polymerase into revealing the sequence•The traditional method – Sanger sequencing – based on “terminator” bases – prevent the polymerase from extending the DNA•Sanger sequencing is essentially PCR + terminator bases• Other methods “spy” on the polymerase as it incorporates nucleotidesCMSC 423 - Fall 2008 7Sanger sequencingSanger, F, Coulson AR. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J.Mol.Biol. 94 (1975)pictures from http://www.uvm.edu/~cgep/Education/Sequence.htmlAGATTATCTAACAGCTACCCTTCCATCATCTAATAGATGGCAGGATCTAATTTCTAATTATCTAATTAGTCTAATTAGATCTAATTAGATCMSC 423 - Fall 2008 8The future of sequencing• Single molecule sequencing - current technology requires many copiesof DNA being sequenced - requires DNA amplification• Massively-parallel sequencing - 100k sequencing reactions occuring atthe same timeSequencing by synthesis Micro-fluidicshttp://www.genetics.ucla.edu/sequencing/pyro.phphttp://www.usgenomics.comAGATTATCTAACAGCTACCCTTCCATCATCTAATAGACMSC 423 - Fall 2008 9The future of sequencing• 454 Life Sciences – approx. 60 Mbp in 200 bp reads / 4 hr run•Solexa Ltd. – approx. 2 Gbp in 30-40 bp reads / 3 day run• ABI SOLiD – 35bp reads – 2 Gbp•Helicos – single molecule sequencing• etc.Massively parallel sequencing• each spot is a molecule or amplified from onemolecule• image processing used to track moleculesduring sequencing by synthesis• often micro-fluidics/lab-on-a-chip
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