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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