Biology Reference
In-Depth Information
Hybridization Selection
Bringing down error rates of microarray oligonucleotide synthesis would involve
substantial efforts. Microarrays typically produce oligonucleotides with lower product
quality, lower product yields, and higher complexity than other sources. Tian et al
.
used
parallel amplification and purification of oligonucleotides from complex oligonucleotide
mixtures using microfluidics to allow for a 2~5-fold increase in quality over PAGE purified
microarray derived oligonucleotides and a 10-fold increase in quality over unpurified
microarray synthesized oligonucleotides.
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This was accomplished using generic
amplification primer sequences for the amplification of all oligonucleotides in a single
PCR reaction. The low yields of microchip-derived oligonucleotides are simply insufficient
for gene assembly without amplification. Additionally, distinct subsets of primer sequences
allow for the fractioning of the oligonucleotide pool into subpools. With a concentrated,
highly complex oligonucleotide starting pool, the potential for undesired cross-
hybridization becomes an issue. Selective amplification of subsets of oligonucleotides
reduces this cross-hybridization.
The difficulty with restriction digested double-stranded microchip oligonucleotides is
that after denaturation, matched antisense oligonucleotides will compete with designated
overlapping oligonucleotides, reducing assembly efficiency. Tian et al. avoided this problem
by enriching single-stranded oligonucleotides by hybridization to antisense selection
oligonucleotides and then eluting them. This was also a purification process, which
allowed for selection against oligonucleotides with errors. These oligonucleotides produce
mismatches during hybridization and elute at a lower temperature. Using selective
amplification and purification via hybridization enabled the synthesis of 21 genes of the
E. coli
30 S ribosomal subunit cluster from a single pool.
In a similar approach, Borovkov et al. combined hybridization-based oligo selection
with parallel amplification.
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Erroneous oligos were eluted and excluded from assembly. By
assembling from unpurified pools of microarray oligos, the authors eliminated purification
steps, reducing assembly costs 100-fold. This approach allows for the robust use of
inexpensive, but imperfect microarray oligonucleotides for gene synthesis.
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Mismatch Recognition
Besides using hybridization selection or improved chemistries with reduced depurination,
a general approach for eliminating errors in gene synthesis is to use DNA mismatch
recognition proteins or enzymes.
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Mismatches are small single-strand loops that
are generated by hybridization between correct and incorrect sequences. There are two
categories of enzymes for mismatch removal: the mismatch binding proteins and the
mismatch cleaving enzymes.
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Mismatch binding proteins like MutS selectively bind to mismatch sites. Incorrect sequences
can be excluded by the removal of protein
DNA complexes with gel-shift assay or affinity
columns. Smith and Modrich first reported the use of MutHLS to remove mutant
sequences.
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MutL couples MutH endonuclease to the MutS bound site, leading to MutH
mediated cleavage. The cleaved heteroduplex was then removed by gel electrophoresis. Using
MutS from
Thermus aquaticus
, Carr et al. established a method that reduced error rate to 1 per
10 kb in two cycles.
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In order to tolerate more errors in longer starting DNA fragments, MutS
treatment can be applied to smaller constituent fragments. In a process called consensus
shuffling, the longer fragment is cleaved into overlapping fragments using restriction
endonucleases. Using column filtration, immobilized MutS can bind to and filter mismatch
fragments. Eluted fragments are then reassembled with PCR. Two iterations improved the
error rate 3.5-fold to 1 per 3.5 kb.
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Because erroneous gene fragments are eliminated in
mismatch filtration, enough initial fragments must exist in the pool to survive MutS binding.
This may prevent mismatch binding from being applied to low-quality and low-yield
