In 1996, David Deamer and Daniel Branton’s labs at UC Santa Cruz and Harvard collaborated on a paper showing that when an electric current runs through a nanopore, passing purine (A and G) and pyrimidine (T and C) DNA bases through the nanopore disrupted the current to different degrees. While the technique couldn’t yet discriminate between all four bases, the general idea for a new single-molecule sequencing method was there.
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3Holley’s method was distinct from Sanger’s. He began by isolating the alanine tRNA molecules and then cutting them into shorter pieces of unequal lengths, using enzymes. Then, he separated each fragment by size using column chromatography and ran various chemical techniques to figure out the sequence of each piece. Finally, he “aligned” these fragments by finding overlaps between them, thus reconstructing the full, 77-nucleotide strand.
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