Biology Reference
In-Depth Information
anhydrous acetonitrile to remove these reagents,
and then purged with argon to remove
the remaining acetonitrile.
DMTO
DMTO
1) Deprotection
The cyclic addition of additional monomers to the
existing oligonucleotide chain occurs in four steps:
deprotection, activation/coupling, capping, and
oxidation (
Fig. 1.1
). Each additional nucleoside
which is added to the growing chain has a
5
0
DMT protection group. This assembly is called a
phosphoramidite. The four-step phosphoramidite
chemistry is the method of choice for most
commercial DNA synthesizers because the yields
are more accurate and homogeneous than other
methods.
4
First, a strong acid is used to de-block
the 5
0
-
O
-4,4
0
-dimethoxytrityl (DMT) group,
removing the protecting group from the nucleotide
chain and exposing a reactive OH group.
In the next step, 1H-tetrazole and the dissolved
phosphoramidite are simultaneously added
to the column. Tetrazole, a weak acid, protonates
the trivalent phosphorus on the 3
0
-end of the
monomer. This results in a slow displacement
of the secondary amine and formation of a highly reactive tetrazolide that then immediately
couples with the OH group. At this point, the added phosphoramidite is coupled to the
existing chain. Uncoupled 5
0
-OH groups are blocked by an acylating capping reagent,
usually acetic anhydride, to minimize deletion products. Finally, the unstable phosphite
triester internucleotide linkage between nucleotides is oxidized to a more stable pentavalent
phosphotriester. The end results of this process are oligonucleotide strands that are bound
to beads. Each phosphate bond contains a methyl group, which can be removed by chemical
treatment in the reaction column. The 5
0
terminus of the last nucleotide can be deprotected
through detritylation of the DMT group, and phosphorylated by T4 kinase. DNA strands
can also then be cleaved from the spacer linker off the solid support.
O
I
P
O
O
HO
O
NC
4)Oxidation
DMTO
2)Coupling
DMTO
OO
I
NC
N(iPr)
2
OO
P
3)Capping
O
H
3
CC
O
O
NC
FIGURE 1.1
The solid-phase, four-step
oligodeoxynucleotide
synthesis cycle.
4
The described four-step synthesis procedure has been the basis of fully automated DNA
synthesizers with up to 1536 sequence throughputs.
5
Throughput evolved from 2
4
individual sequences in initial synthesizers manufactured by Applied Biosystems, to 96 well
plates in 1995.
6
Lashkari et al. used computer-controlled solenoid valves to deliver
bulk reagents through Teflon tubes into a microwell plate. Since then, parallel synthesis
using multiplexed reagent delivery lines has allowed for synthesis in other microwell
plate formats.
Optimizations in reaction chemistry include a two-step cycle synthesis, which reduces
costs by eliminating several reagents.
7
A peroxy anion is used as a nucleophile to remove a
5
0
-carbonate and oxidize the internucleotide phosphite triester. Deprotection with peroxy
anion under mildly basic conditions can eliminate depurination, a side reaction that leads
to mutations in synthetic DNA. If further developed, the two-step synthesis process can
make oligonucleotide synthesis simpler, and consequently more robust.
Microarray Oligonucleotide Pool Synthesis
The major costs for gene synthesis are attributed to oligonucleotide synthesis, sequence
verification, and labor for processing steps. Microarray-enabled oligonucleotide pool
synthesis effectively tackles oligonucleotide costs. Since their inception in 1995, microarrays
have dramatically revolutionized genomics with massive parallelism and automation.
