Instead of promoting irreversible primer extension like the Sanger method, the reversible chain terminators method uses a cyclic method that consists of nucleotide incorporation, fluorescence imaging and cleavage. The figure below shows a modified nucleotide with a cleavable dye and reversible blocking group. Once the blocking group is removed, a new nucleotide may come in.
The steps for such a process can be outlined as follows:
Have four dNTP's, each with a different fluorescent marking. These markings should not interfere with base pairing or phosphodiester bond formation.
Each dNTP should terminate DNA elongation temporarily with a blocking group on the 3' carbon of the sugar moiety.
Upon each cycle, have just one dNTP bind to the elongating strand and emit a fluorescent dye color.
Depending on the color emitted, record the particular nucleotide.
Cleave the blocking group and fluorescent dye with a palladium-catalyst.
Restore a 3' hydroxyl so that the growing strand can now elongate.
Repeat from step 1.
There are some limitations to this method which include:
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