Funding limitations prevented Nyren from dedicating much time to the project at first, but in 1993, he was finally able to publish a proof-of-principle. His technique began by mixing the template DNA with a primer, a single dNTP, and three enzymes: the familiar DNA polymerase plus the light cascade enzymes, ATP sulfurylase and firefly luciferase.6 If the dNTP was incorporated into a strand of DNA, PPi would be produced in the chemical reaction. ATP sulfurylase could then convert the PPi into ATP, which would provide energy for the luciferase enzyme, producing light. Thus, it was possible to determine each base in the sequence by cycling through the dNTPs one at a time until light was detected, and then washing extra nucleotides out between each step. By literally rinsing and repeating, the sequence could be recorded one letter at a time without the use of any gels, which often took hours to run and were difficult to automate.
warning rather than error, at least for now.[1]
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