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


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.


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.

Learn to be a Pythonista!

Python Playground

Learn to be a Pythonista! Try Python

Python Playground is a collection of fun programming projects that will inspire you to new heights. You'll manipulate images, build simulations, and interact with hardware using Arduino & Raspberry Pi. With each project, you'll get familiarized with leveraging external libraries for specialized tasks, breaking problems into smaller, solvable pieces, and translating algorithms into code.

$ Check price
29.9529.95Amazon 4 logo(14+ reviews)

More Python resources

Become a Bioinformatics Whiz!

Bioinformatics Data Skills

Become a Bioinformatics Whiz! Try Bioinformatics

Learn the best practices used by academic and industry professionals. Bioinformatics Data Skills give a great overview to the Linux Command Line, Github, and other essential tools used in the trade. This book bridges the gap between knowing a few programming languages and being able to utilize the tools to analyze large amounts of biological data.

$ Check price
49.9949.99Amazon 4.5 logo(7+ reviews)

More Bioinformatics resources