05. Reversible chain terminators

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.

Reversible 3'-O-blocked reversible terminator
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Procedure

The steps for such a process can be outlined as follows:

  1. Have four dNTP's, each with a different fluorescent marking. These markings should not interfere with base pairing or phosphodiester bond formation.
  2. Each dNTP should terminate DNA elongation temporarily with a blocking group on the 3' carbon of the sugar moiety.
  3. Upon each cycle, have just one dNTP bind to the elongating strand and emit a fluorescent dye color.
  4. Depending on the color emitted, record the particular nucleotide.
  5. Cleave the blocking group and fluorescent dye with a palladium-catalyst.
  6. Restore a 3' hydroxyl so that the growing strand can now elongate.
  7. Repeat from step 1.

Cons

There are some limitations to this method which include:

  • Incomplete cleavage of blocking groups.
  • Difficulties incorporating fluorescent nucleotides.
  • Only up to 36 nucleotides per run.

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