Dideoxynucleotide chain termination oligonucleotides and their application

A 3'-chain terminator or end blocker oligonucleotide modifications terminate DNA synthesis and block ligation at the 5'-terminus.

Because of their unique properties, several molecular biology methods utilize dideoxynucleotides as chain terminators or blockers. The dideoxynucleotide chain-terminating method uses deoxyribonucleotides lacking a hydroxyl group (OH) at the 3'-positions of the ribose sugar. Oligonucleotides modified with chain terminator or end blocker dideoxynucleotides at the 3'-end block ligation or prevent polymerase extension from the 3'-terminus. For oligonucleotides modified with a dideoxyribonucleotide, a phosphodiester bond cannot form with a 5'-hydrogen resulting in a chain elongation stop.

The lack of a 3'-OH group on the ribose sugar makes dideoxynucleotides a valuable tool for the following applications:

1. DNA sequencing

The Sanger sequencing method, a widely used technique to determine the sequence of DNA molecules, uses dideoxynucleotides.

2. Site-directed mutagenesis

Dideoxynucleotides help to create mutations in specific regions of DNA. This technique incorporates a dideoxynucleotide into the growing DNA strand during replication, which terminates the chain and introduces a mutation.

3. In vitro transcription

Dideoxynucleotides can terminate RNA synthesis during in vitro transcription producing RNA molecules that have a defined 5'- and 3'-end.

4. Primer extension

In primer extension assays, dideoxynucleotides allow the determination of the position of specific nucleotides in a DNA or RNA molecule. In this technique, a primer is annealed to the target molecule, and DNA polymerase extends the primer in the presence of dideoxynucleotides. Incorporating a dideoxynucleotide at a specific position terminates the extension reaction, indicating the position of the nucleotide of interest

5. Biotinylated dideoxynucleotides for mass spectrometry analysis

Biotinylated dideoxynucleotides (biotin‐ddNTPs) enable single base extension reactions for multiplex genotyping by mass spectrometry (MS) (Kim et al. 2002). In this method, oligonucleotide primers of different molecular weights specific to polymorphic sites in a DNA template are extended with biotin‐ddNTPs by DNA polymerase to generate 3'‐biotinylated DNA products. Unextended primers and other components in the reaction are washed away, but streptavidin‐coated solid phase magnetic beads capture the resulting extension products. The extension products are released from the solid phase and analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight MS.

6. End labeling of oligonucleotides

The template-independent polymerase "terminal deoxynucleotidyl transferase" (TdT, or terminal transferase) enables selective enzymatic labeling of oligonucleotides at the 3'-terminal end.TdT catalyzes the addition of deoxynucleotides and dideoxynucleotides (ddNTPs) to the 3′-hydroxyl termini of a DNA molecule in the presence of its cofactor cobalt (Co2+). Using ddNTPs for the reaction limits the incorporation of nucleotides to just one nucleotide.

Incorporating [α-32P]ddATP or biotin-, digoxigenin (DIG)-, or fluorescein-ddUTP at the 3'-end enables enzymatic labeling of oligonucleotides with a radioactive tracer, biotin, digoxigenin, or fluorescein. Similarly, the enzyme can add ddNTPs modified with other functional groups to the 3'-end of oligonucleotides as long as they fit into the enzyme's catalytic pocket. Since none of these molecules carry a 3'-hydroxyl group, no other nucleic acid can be incorporated. 

Termination of DNA synthesis by incorporation of ddNTPs.

DNA polymerase-catalyzed esterification of the normal dNTP with the 3'-terminal nucleotide of DNA. The reaction extends the length of the primer by a single nucleotide. The reaction continues until all bases in a template strand are paired with the newly synthesized strand.

DNA polymerase-catalyzed esterification of a ddNTP with the 3'-terminal nucleotide of DNA. The ddNTP lacks a 3'-hydroxyl residue. Incorporating a ddNTP into the growing chain by DNA polymerase acts as a chain terminator because the 3’-hydroxyl group required to form a further 5'->3' phosphodiester bond is not available for the reaction. 

The Sanger DNA sequencing method uses dideoxynucleotides as chain-elongating inhibitors or chain terminators of DNA polymerase. The abbreviation of dideoxynucleotides is ddNTPs (ddGTP, ddATP, ddTTP, or ddCTP). Because the ribose's 2'- and 3'-position do not contain hydroxyl groups, dideoxynucleotides are also known as 2', 3'-dideoxynucleotides.

Dideoxynucleotides can be labeled with a radioactive or nonradioactive tag to visualize fragments containing ddNTPs.

The earlier Taq polymerases used were deficient in two respects:

(i) During sequencing, the enzymes incorporate each of the four dideoxynucleoside 5'-triphosphates (ddNTPs) at widely different rates (ddGTP, for example, was incorporated ten times faster than the other three ddNTPs), and 

(ii) The enzymes exhibited uneven band-intensity or peak-height patterns in radio-labeled or dye-labeled DNA sequence profiles; therefore, Li et al., in 1999, created Taq polymerase variants with improved biotechnological specificities converting these polymerases to functional tools:

[1] With a better extension of guanine (G) bases, and

[2] a more consistent band-intensity pattern allowing for more accurate sequencing results.

As a result, during genome sequencing, using Taq DNA polymerases mutated at position 660 helped limit errors and reduce the requirement for redundancy, thereby decreasing cost and labor.


Green MR, Sambrook J. Labeling the 3' Termini of Oligonucleotides Using Terminal Deoxynucleotidyl Transferase. Cold Spring Harb Protoc. 2021 Aug 2;2021(8). [CSH Protocols]

Sobin Kim, John R. Edwards, Liyong Deng, Wendy Chung, Jingyue Ju, Solid phase capturable dideoxynucleotides for multiplex genotyping using mass spectrometry, Nucleic Acids Research, Volume 30, Issue 16, 1 August 2002, Page e85. [NAR]

Li Y, Mitaxov V, Waksman G. Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation. Proc Natl Acad Sci U S A. 1999 Aug 17;96(17):9491-6. [

Sanger, F.
 (8 December 1980). "Determination of Nucleotide Sequences in DNA (Nobel lecture)".

Terminal Deoxynucleotidyl Transferase


Bio-Synthesis provides oligonucleotides modified with chain terminator modifications as well as a full spectrum of bio-conjugation services including high quality custom oligonucleotide modification services, back-bone modifications, conjugation to fatty acids and lipids, cholesterol, tocopherol, peptides as well as biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.

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