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University of Toronto St. George
Molecular Genetics and Microbiology
William Navarre

18 Sequencingprocedures to determine the exact order of nucleotides in the DNA fragment Maxim and Gilbert Methodimproved throughput see sequence from where cleaved But Sanger method is the standardcurrently Method is based on DNA polymerase dependent synthesis of a complementary DNA strand in presence of natural 2 deoxynucleotides and 2 3 dideoxynucleotideso Serve as nonreversible synthesis terminatorso DNA synthesis reaction randomly terminated whenever a ddNTP is added to the growing oligonucelotide chain Steps to sequencing bacterial genome Whole genome shotgun sequencing methodo DNA randomly sheared into small fragments that are ligated into plasmids o Thousands of plasmids are generated each of which has some random fragment of the genome you want to sequenceo This library of plasmids are put into E coli so that they may be purified in amounts sufficient for sequencingo Each colony of E coli has different plasmido Plasmids are isolated from the thousands of E coli clones each harbouring a plasmid with a different random segment of the genomeo Determine five h
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