Biology Reference
In-Depth Information
1
CHAPTER
New Tools for Cost-Effective
DNA Synthesis
Nicholas Tang
1
, Siying Ma
1
and Jingdong Tian
1,2
1
Duke University, Durham, NC, USA
2
Chinese Academy of Sciences, Tianjin, China
INTRODUCTION
DNA synthesis is a powerful enabling technology and yet a limiting step in synthetic
biology. Decreasing costs in DNA synthesis will open new frontiers and project concepts
that would not be feasible to most scientists at current levels. The cost of column
synthesized oligonucleotides has dropped 10-fold over the past 15 years,
1
and is currently
around USD0.08
0.2 per nucleotide.
2
Gene synthesis is a more expensive process, and
involves generating longer DNA constructs from overlapping oligonucleotides. In 2000, the
market cost of gene synthesis was approximately USD10 per base.
3
Since then, prices have
dropped nearly 50-fold in 10 years, to as low as USD0.2 per base pair, with average error
rates of about 1 in 300
3
600 bases.
1,2
Prices continue to drop by a factor of 1.5 per year, in
s law.
1
By 2005, there existed 39 gene synthesis vendors
worldwide, and the number has increased since then. In the past couple of years, more
companies have begun to emerge to take on the challenge of adapting new synthesis
technologies for the market. It would be interesting to see dramatic changes to the gene
synthesis market in the near future.
a manner akin to Moore
'
OLIGONUCLEOTIDE SYNTHESIS
Column Oligonucleotide Synthesis
Chemical DNA synthesis can be used for applications as common as primer, linker,
or probe synthesis. Here we discuss oligonucleotide synthesis for the application of gene
and genome synthesis. Chemical assembly of DNA using programmable synthesizers is now
a routine procedure. The most common and reliable system for chemical synthesis involves
synthesizing individual oligonucleotides in small columns. A series of valves and pumps
introduce the correct nucleotide monomers and reagents required for growing oligomers
of DNA in a stepwise manner. Chemical oligodeoxynucleotide synthesis is different
from enzymatic DNA synthesis in living cells in that it is a cyclical process that elongates
nucleotides from the 3
0
to the 5
0
-end. The starting complex for chemical synthesis of
DNA consists of an initial acid-activated nucleoside phosphoramidite tethered with a spacer
to a solid-support controlled pore glass (CPG) or polystyrene (PS) bead. The advent of
solid-phase DNA synthesis made automation possible by eliminating purification steps
to remove intermediates or unreacted reagents. The column is simply rinsed with
