Lecture 4 - Genome Sequencing & Assembly (Chapt 4).pptx

Lecture 4 - Genome Sequencing & Assembly (Chapt 4).pptx...

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Lecture 6 Sequencing Sanger method Genome sequencing with Sanger
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Stratton Nature 45: 719 , 2009 Evolution of DNA sequencing technologies - 1980 to present day DNA SEQUENCING & ASSEMBLY 3000 x 10 9 bp per day (NovaSeq6000, 2017)
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… first protein coding gene sequenced from RNA bacteriophage MS2 … its genome was also the 1 st sequenced First attempts at sequencing (mid 1960 – mid 1970s) - RNA only (tRNA ssRNA genomes) easy to isolate large amounts single strand determined through enzymatic digest and chemical degradation Yeast alanine tRNA 77 residues MS2 bacteriophage Sequence Hallry in 1 dsDNA was too complex to sequence - Coat protein 510 nt - Walter Fiers 1972 - DNA gel electrophoresis helped him do this - Genome = 3569 nt - Fiers lab 1976 - Digest rna, separate by electrophoresis, then degradation to come up w/ a
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… but it stops when dideoxynucleotide is incorporated Sanger chain termination method (Fred Sanger, 1977) - enzymatic synthesis of DNA strand complementary to “template” of interest Nobel Prizes: Sanger 1958 (protein structure) 1980 (DNA sequencing) - Took advantage of dna replication to be able to sequence DNA - Slightly modified DNAP from E.coli – used Klenow fragment - Cut off domain that did 5’ to 3’ exonuclease activity in DNAP
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Sanger chain termination method Sample sequence ddATP + 4 dNTPs ddCTP + 4 dNTPs ddGTP + 4 dNTPs ddTTP + 4 dNTPS 4 parallel sets of reactions - Balance - Too - Too - Gel elect - Wer - No - Use - Diffi - Ran - P or - Can read
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Interview of Dr. Sanger Sanger chain termination method (Fred Sanger, 1977) revolutionized molecular biology This was THE standard method for >20 years !! “For all of Sanger sequencing’s high cost, it remains the benchmark for accuracy .” Hayden Nature 507:294, 2014 first DNA-based genome phiX174 5386 bp human mitochondrial DNA 16 kb bacteriophage λ 48 kb Initial limitations: - Sensitivity -
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