Biology Reference
In-Depth Information
65
C.
Because the ligation reaction is linear, the full construct is designed with flanking primer
sequences so that PCR can amplify the construct.
are first heat denatured, and then cooled for annealing and ligation at 50
PCR-based assembly, otherwise known as polymerase cycling assembly (PCA), remains
one of the most cost-effective gene assembly methods.
51,52
In a two-step procedure,
partially overlapping oligonucleotides are designed to span the whole sequence.
53
Because
gaps between oligonucleotides on the same strand are allowed, the number of starting
oligonucleotides is fewer than that required for ligation-based assembly. A PCR reaction
is carried out so that overlapping oligonucleotides anneal and extend. The resulting
double-stranded construct can serve as the template for the PCR amplification reaction. In a
single-step procedure, the amplification primers are included with the oligonucleotides for
a combined assembly and amplification reaction.
54,55
Although extra cycles may be needed,
this procedure is more easily multiplexed than assembly with multiple steps.
53
Although
PCR-based methods are efficient, constructs involving repetitive sequences or secondary
structures may have difficulties during PCR, and thus can better be assembled with
ligase-based assembly.
56
There are a number of related overlapping extension techniques for sequence-independent
assembly. For the sequence-independent assembly of circular double-stranded constructs
or plasmids, circular polymerase extension cloning (CPEC)
57
can successfully assemble not
only multiple-gene constructs, but also combinatorial sequence libraries.
19
In a single-step
reaction, overlapping oligonucleotides are assembled and circularized. Other approaches
suitable for plasmid construction include the In-Fusion commercial kit from Clontech,
58
Uracil-specific excision reagent (USER)
59
and Sequence- and Ligation-Independent Cloning
(SLIC).
60
Errors may arise during the assembly of large constructs. Gibson isothermal or
'
assembly avoids length-dependent errors with the T5 DNA
polymerase, allowing for assembly of genome length constructs,
61
such as a 16.3-kb
mitochondrial genome.
62
chewback and anneal
'
10
Rather than cleaving oligonucleotides from microchips, Quan et al. uses an approach
involving isothermal nicking and strand displacement amplification (nSDA) of
immobilized microarray oligonucleotides.
19
This simultaneously amplifies and releases
oligonucleotides, which are then PCA assembled into 1-kb constructs. A microfluidic
system serves to integrate synthesis, amplification and assembly (
Fig. 1.3
).
19,63
By performing all steps on chip, high-throughput assembly can be easily coupled
with downstream reactions.
Although high-fidelity DNA microchips can hold up to a million unique oligonucleotides,
they are difficult to scale. Microarray oligonucleotide pools are highly complex, and become
problematic in complications like potential cross-hybridization between assembled
fragments. More successful scaling will lower the cost of high-quality gene synthesis.
To address this issue, Kosuri et al. combined selective oligonucleotide pool amplification,
optimized gene assembly, and enzymatic error correction.
12
The authors cleaved
microchip-synthesized oligonucleotides. These oligonucleotides were synthesized with
flanking amplification primer annealing sites corresponding to several subpools. A quarter-
million specific amplification primers were then used to selectively PCR amplify specific
subpools of oligonucleotides from the original complex background of oligonucleotides.
The flanking amplification primer sequences were then cleaved with restriction enzymes,
which allows for seamless subsequent PCR assembly into gene constructs. The authors
tested the system on 47 genes encoding for proteins and antibodies, including 40 error-free
single-chain antibody genes that had previously been shown to be difficult to synthesize
due to high GC content and repetitive sequences. The assembly optimization and enzymatic
treatment allowed for accurate and low-cost synthesis, bringing costs down to an estimated
USD0.01/bp.
