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
this

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.

Take your Linux skills to the next level!

Linux for Beginners

Take your Linux skills to the next level! Try Linux & UNIX

Linux for Beginners doesn't make any assumptions about your background or knowledge of Linux. You need no prior knowledge to benefit from this book. You will be guided step by step using a logical and systematic approach. As new concepts, commands, or jargon are encountered they are explained in plain language, making it easy for anyone to understand.

$ Check price
24.9924.99Amazon 4.5 logo(101+ reviews)

More Linux & UNIX resources

Learn to be a Pythonista!

Python Programming

Learn to be a Pythonista! Try Python

This book is designed to be used as the primary textbook in a college-level first course in computing. It takes a fairly traditional approach, emphasizing problem solving, design, and programming as the core skills of computer science. However, these ideas are illustrated using a non-traditional language, namely Python.

$ Check price
45.9945.99Amazon 4.5 logo(211+ reviews)

More Python resources

Ad