Why are oligos synthesized 3′−5′?

The cell may synthesize DNA from 5′−3′, but synthetic oligos are made much more efficiently in the 3′−5′ direction. Learn why in a brief history on in vitro synthesis of oligos.

Jan 10, 2012

Revised/updated Jun 6, 2017

In nature, DNA is formed in the 5′–3′ direction. Early efforts in DNA synthesis were based on biological synthesis, and thus the first synthetic oligonucleotides were produced in the 5′–3′ direction [1]. Har Gobind Khorana, a University of Wisconsin biochemist who won the 1968 Nobel Prize in Physiology or Medicine [2], led the group that developed the early 5′–3′ synthesis technique using a polystyrene solid support and three different protecting groups.

Though this technique led to important breakthroughs, it was eventually replaced in the 1980s by much a more efficient synthesis method using phosphoramidite monomers (phosphoramidites are nucleotides with protection groups which are removed after synthesis) [3]. The growing oligonucleotide is connected to the solid support, a controlled pore glass bead via the 3′ carbon, and thus synthesis proceeds in the 3′–5′ direction.


  1. Integrated DNA Technologies. (2011) Chemical synthesis of oligonucleotides. [Online] Coralville, Integrated DNA Technologies. [Accessed 16 Nov, 2017].
  3. Beaucage SL and Caruthers MH (1981) Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Letters 22: 1859–1862.