Synthetic Biology. Tools and Applications

331 Pages·2013·13.18 MB·English

by  Huimin Zhao (Eds.)

(cid:1)
1
CHAPTER
New Tools for Cost-Effective
DNA Synthesis
Nicholas Tang1, Siying Ma1 and Jingdong Tian1,2
1Duke University, Durham, NC, USA
2Chinese Academy of Sciences, Tianjin, China
INTRODUCTION
DNAsynthesis is apowerfulenablingtechnology and yetalimiting step in synthetic
biology. Decreasingcostsin DNA synthesis will opennew frontiers andproject concepts
that would not be feasible tomost scientists at current levels. The costof column
synthesized oligonucleotides has dropped 10-foldover the past15 years,1 and is currently
around USD0.08(cid:1)0.2 per nucleotide.2 Gene synthesis is amore expensive process, and
3
involves generating longer DNAconstructs from overlapping oligonucleotides. In 2000, the
marketcostof gene synthesis was approximatelyUSD10 per base.3Sincethen,prices have
dropped nearly 50-fold in10 years, to as low as USD0.2 perbasepair, with average error
rates of about 1 in300(cid:1)600 bases.1,2Prices continue todropby afactorof 1.5 per year, in
amanner akin to Moore’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 challengeof adapting new synthesis
technologies for the market.It would be interesting to see dramatic changes to the gene
synthesis marketin the near future.
OLIGONUCLEOTIDE SYNTHESIS
Column Oligonucleotide Synthesis
Chemical DNA synthesis can be used for applications as commonas primer, linker,
or probe synthesis. Here we discuss oligonucleotide synthesis for the application ofgene
and genome synthesis. Chemical assemblyofDNAusingprogrammable synthesizers is now
aroutine procedure. The most commonandreliablesystem for chemical synthesis involves
synthesizingindividual oligonucleotidesin small columns. A series ofvalvesand pumps
introduce the correct nucleotide monomers and reagents required for growing oligomers
of DNA in astepwisemanner. Chemical oligodeoxynucleotide synthesis isdifferent
from enzymatic DNA synthesis in living cells in that it is acyclical process that elongates
nucleotides from the 30 tothe 50-end. The startingcomplexfor chemical synthesis of
DNAconsists of aninitial acid-activated nucleoside phosphoramidite tethered with aspacer
to asolid-support controlledporeglass (CPG) or polystyrene (PS) bead. The advent of
solid-phaseDNA synthesis made automation possible by eliminating purification steps
to remove intermediates or unreacted reagents. Thecolumn is simply rinsed with
SyntheticBiology.DOI:http://dx.doi.org/10.1016/B978-0-12-394430-6.00001-7
©2013ElsevierInc.Allrightsreserved.
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Synthesis and Engineering Tools in Synthetic Biology
DMTO anhydrous acetonitrile toremove thesereagents,
and thenpurgedwithargon to remove
DMTO
the remaining acetonitrile.
1) Deprotection
Thecyclicadditionofadditionalmonomerstothe
O existingoligonucleotidechainoccursinfoursteps:
I O
O P HO deprotection,activation/coupling,capping,and
O oxidation(Fig.1.1).Eachadditionalnucleoside
NC
whichisaddedtothegrowingchainhasa
50DMTprotectiongroup.Thisassemblyiscalleda
4)Oxidation phosphoramidite.Thefour-stepphosphoramidite
DMTO
chemistryisthemethodofchoiceformost
commercialDNAsynthesizersbecausetheyields
2)Coupling
DMTO
aremoreaccurateandhomogeneousthanother
O O methods.4First,astrongacidisusedtode-block
NC P
I the50-O-4,40-dimethoxytrityl(DMT)group,
N(iPr)2
O O 3)Capping removingtheprotectinggroupfromthenucleotide
P chainandexposingareactiveOHgroup.
I O
O Inthenextstep,1H-tetrazoleandthedissolved
H3CC O
NC phosphoramiditearesimultaneouslyadded
tothecolumn.Tetrazole,aweakacid,protonates
thetrivalentphosphorusonthe30-endofthe
monomer.Thisresultsinaslowdisplacement
FIGURE 1.1 ofthesecondaryamineandformationofahighlyreactivetetrazolidethatthenimmediately
Thesolid-phase,four-step coupleswiththeOHgroup.Atthispoint,theaddedphosphoramiditeiscoupledtothe
oligodeoxynucleotide existingchain.Uncoupled50-OHgroupsareblockedbyanacylatingcappingreagent,
synthesiscycle. usuallyaceticanhydride,tominimizedeletionproducts.Finally,theunstablephosphite
4
triesterinternucleotidelinkagebetweennucleotidesisoxidizedtoamorestablepentavalent
phosphotriester.Theendresultsofthisprocessareoligonucleotidestrandsthatarebound
tobeads.Eachphosphatebondcontainsamethylgroup,whichcanberemovedbychemical
treatmentinthereactioncolumn.The50terminusofthelastnucleotidecanbedeprotected
throughdetritylationoftheDMTgroup,andphosphorylatedbyT4kinase.DNAstrands
canalsothenbecleavedfromthespacerlinkeroffthesolidsupport.
Thedescribed four-step synthesis procedure has been the basisof fully automatedDNA
synthesizers with up to 1536 sequence throughputs.5Throughputevolvedfrom 2(cid:1)4
individual sequencesin initial synthesizers manufactured by Applied Biosystems, to96 well
plates in1995.6 Lashkari et al.usedcomputer-controlled solenoidvalvestodeliver
bulk reagents through Teflontubesinto amicrowellplate.Sincethen,parallelsynthesis
using multiplexed reagentdelivery lines hasallowed for synthesis in other microwell
plate formats.
Optimizations in reaction chemistry include atwo-stepcyclesynthesis, whichreduces
costs by eliminating severalreagents.7 Aperoxyanion is used as anucleophile to remove a
50-carbonate andoxidize the internucleotide phosphitetriester. Deprotection with peroxy
anion under mildly basic conditions can eliminate depurination, aside reaction thatleads
tomutations in syntheticDNA. Iffurtherdeveloped, the two-step synthesis process can
make oligonucleotide synthesis simpler, and consequently more robust.
Microarray Oligonucleotide Pool Synthesis
Themajorcostsfor gene synthesis are attributed to oligonucleotide synthesis, sequence
verification, andlaborfor processing steps.Microarray-enabled oligonucleotide pool
synthesis effectively tackles oligonucleotide costs. Since their inception in 1995, microarrays
have dramaticallyrevolutionized genomics withmassiveparallelismand automation.
CHAPTER 1
New Tools for Cost-Effective DNA Synthesis
Microarrays are 2D solid-phasearraysusedto assay or screen biological materials like
nucleic acids, proteins, or cells. Oligonucleotide arrays can be usedfor avariety ofdesigns,
including gene expression screens,SNP genotyping, comparative genomic hybridization
(CGH),tiling, ChIP-on-chip, microRNA, resequencing,and aptamer screening.8More
relevantly, oligonucleotide microarrays can offer significant reductions inDNA synthesis
cost due to their dense and massively parallel feature designs. For example, reducing costs
by scaling down reagent volumesin resin-based synthesis is restrictedtodecreasing the
diameter of capillaries. Simplyeliminating the rinsing of lines in instrumentation makes
at leasta10-fold reduction incost.6This is because acetonitrile is often used in high
volumes for rinsinganddissolving reagents, making it one of the most expensive bulk
reagents.9 Cost for reagents in acustom inkjetmicroarray slideis less than USD50 per slide,
due to the low volumes of phosphoramiditeand tetrazole necessaryfor the miniaturized
platform.10 Microarray synthesis not only lowerscosts, but also makes synthesis more
environmentally friendly. High-density arrays can offer 104(cid:1)106 unique oligonucleotides,
and can reduce costs by at least anorder ofmagnitude.11,12 Theprice for 3912 90-mers
from LC Sciences is about USD1000; the Agilent 55k chip,BUSD7000, which translates to
USD0.0025 per base.13These costsdo not include downstream costs for gene assembly.
The variety in technologies andtechniques for microarray-based oligonucleotide synthesis
is expected tooffer reductions in cost andimprovements in throughput in the comingyears.
In contrast, the cost of column-synthesized oligonucleotideshasremained constant over
the pastsix years and is unlikelyto decrease significantly.14Themicroarray technologies
that exist in the DNA synthesis market include ink-jet printing (Agilent, Protogene),
photosensitive50 deprotection (Nimblegen, Affymetrix, Flexgen), photo-generated acid
deprotection (Atactic/Xeotron/Invitrogen, LCSciences), andelectrolytic acid/base arrays
(Oxamer, Combimatrix/Customarray).
Optimizationsthat havebeen made to microarray synthesis include synthesis on 5
PDMS(poly(dimethylsiloxilane))15and COC (cyclicolefin copolymer)16substrates as
low-cost andflexiblealternatives to glass. Ma and coworkers showed that oxCOC, ahybrid
substrate composed of COC and RF sputtered SiO , takes benefits from both constituents.17
2
COC offerslow density, resistance to organic solvents,highstiffness, andUV transparency,
whileSiO offers useful surface linkers for phosphoramidite chemistry.Furthermore,
2
oxCOC can be manipulated for large-scaleproduction.With soft lithography,aPDMS
stamp can be made to imprint channelsor wells on COC before thin-film deposition
of SiO .18 Thestamps are disposable andcan be createdin anon-cleanroom setting with
2
silicon molds,although the silicon molds must be fabricated viaphotolithography. A study
involving inkjetmicrochip synthesis demonstrated that using a COC chip with patterned
silicafeatures reduced the error rate of synthesized oligonucleotides from one in 200 bases
to one in 600 bases,16whichis equivalent to high-quality column synthesis.
The dominant costsare now enzymatic processing,cloning, andsequencing.12A recent
study by Tian’s groupaddresses the limitations ofprocessing stepsusing an integrated
combination of isothermalnicking, strand displacement amplification (nSDA), and
polymerase cycling assembly (PCA),reducingthe costeven further toUSD0.005 per base
with an error frequency of ,0.2 errors/kb.19Suchreduced cost could make gene and
gene librarysynthesis more widely accessible.
Microfluidic and Fluidic Systems
Microfluidics allows for the controlandmanipulation ofsmallvolumesof liquids.
These features make it particularly useful for a variety of applications like PCR andcell
screening. Morespecifically, microfluidics can decreasethe space andcostrequirements
of DNA synthesis. A recent study reports aprogrammable microfluidic synthesis platform
that can synthesizeB100pmol of each unique oligonucleotide,whichis substantial
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Synthesis and Engineering Tools in Synthetic Biology
enough not to require PCR amplification steps before gene assembly.20The output levels
from this study better match the amountsnecessary for gene assembly.Using microfluidics
for DNA synthesis reducesreagent consumption by 100-fold compared toconventional
column synthesis which generatesfar more oligonucleotides than necessary for gene
assembly.Onthe otherhand,although microarrays can synthesize alarge number
ofoligonucleotides, they produce only 106(cid:1)108 moleculesper spot andyield less than
2fmol. This yield is six orders ofmagnitude lower than thoseof column-synthesized
oligonucleotides,20,21 whichare too low for gene assemblywithout amplification.
Althoughamplification methods havebeenrefined, PCR amplification can introduce errors
andincrease overall labor andprocessing costs. Additionally, there is a100-foldreduction
ofreagentconsumption compared toothersolid-phasesynthesis technologiesthat have less
efficient deblocking steps.20DNA synthesis with microfluidics as performedin this study
can furtheradvancelab-on-a-chip (LOC)or Micro Total AnalysisSystems (μTAS).
However, the 17(cid:1)24 mer length and 1in 153bp error rates couldstillbe refined tobe
suitable for longer gene constructs.
Althoughmicrofluidicsallows for massively parallel microscale DNA synthesis, there are
afewkey technical challenges. One challenge is organization of addressable synthesis units.
Xiao et al.22used soft lithographytosynthesize oligonucleotide arrayson glass surfaces.
During the coupling step ofsynthesis, pre-cast PDMS microstamps transferredamixture
ofnucleoside monomerand tetrazole onto the glass slides surface. Like photomasks in
photolithography, different stamps dictate predefined areas for the couplingof transferred
monomer.20 mers were synthesized with highcoupling efficiency, with astepwise
yield of 97%.
Blair etal.23used365nm wavelength ultraviolet-light-emitting diodes(UV-LEDs) as a
cost-effective alternative light source for addressing anddirecting oligonucleotide synthesis
6 inside glass capillaries. A string of UV-LEDs were positioned along the length of aglass
capillary.Theinside walls werefunctionalizedfor oligonucleotide synthesis using
photolabile 2-nitrophenyl propoxycarbonyl(NPPOC) chemistry.24 The glass capillaries
were designated capillary synthesis cells (CSC) in theirfluidic system. Becausethe spectrum
ofthe UV-LED containedno emission below 360nm, no filters wereneededto prevent
DNA damagefrom shortwavelength radiation. 70 mersweresuccessfully synthesized
andusedfor gene assembly.
Anothertechnical challengeis the chemical resistance of microfluidic substrates.
For example,elastomers are apopular materialfor microfluidic devicesbecause oftheir
fabrication cost and labor benefits over silicon. However, elastomers must be chosenor
modifiedto be chemically resistant to the organic solventsusedin oligonucleotide
synthesis. Popularelastomers like poly(dimethylsiloxane) (PDMS)degrade,swell and clog
incontact with DNA synthesis reagents, whichresults in as high as a90% flow rate drop.25
Moorcroftet al.15circumvented this issue by substituting both the oxidation and
deprotection solvents in conventional DNA synthesis. PDMSmicrochannels were first
molded by asoft lithographyprocess. Then, the surfacewas functionalized by silanizing
with 3-glycidoxypropyltrimethoxysilane (GPTMS)and adding aPEG spacer.21 mers were
synthesized on the PEG-Silane-PDMS chip. The Quake groupusedachemicallyresistant
photocurable perfluoropolyether(PFPE) for theirmicrofluidic synthesis chambers.26To
avoid functionalization, the synthesis chambers weremade ofPFPE, whileoligonucleotide
synthesis was performed on porous silica beads.Thedevicewas usedto synthesize 60pmol
of20 mersoligonucleotideswith60-fold less reagentconsumption than conventional
synthesis.
Anotherchemically resistant substrate is carbon. Carbonoffers superiorheat andchemical
stabilities compared toglass, allowing for robust linkageof oligonucleotide probes.27
CHAPTER 1
New Tools for Cost-Effective DNA Synthesis
Phillips etal. functionalized carbon surfacesfor light-directedoligonucleotide array
synthesis using RF plasma treatmentand9-decene-1-ol. Theauthors usedtwotypes of
carbon as substratesfor DNA synthesis: glassy carbon and CVD diamond.Oligonucleotide
synthesis was thenperformed on thesesurfaceswithphotolabileNPPOC chemistry. Despite
robust oligonucleotide linkage, carbon-based DNA probe arrays28,29 are not yet commonly
used,due to their low-density probe coverage.
Hua andGulari25reported aplatform that integrated apneumatic microvalve array in a
microfluidic system toprecisely control the flow offluidics for parallel oligonucleotide
synthesis. Theycoated the PDMS valve membraneswith paryleneto make themchemically
resistant and compatiblewithaggressive chemical reagents. These valvemembranes were
sandwiched between aPDMS and asilicon layer.ThePDMS layer provided air channels,
whilethe fluid reactionsoccurred on the silicon layer. The pressurized air channels closed
the valvesand restrictedfluidsfrom leaving. Sixteenmultiplexed air channelsweresufficient
to control 12870 reactors. 30 mersweresuccessfully synthesized with astepwise synthesis
yield ofB99.5%.
Photolithography
Some of the earliest efforts tosynthesize oligonucleotides on microarraysinvolved using
physicalphotolithographic masks todirect alight pattern on nucleoside monomers
with photolabileprotecting groups.30,31 Lightpatterns are directed toareas on the array
to remove photolabile protecting groupsfrom the growing oligomers.After deprotection,
aselected phosphoramiditemonomer is spreadover the entire surface andonly the exposed
areas are activated for coupling ofthatparticular phosphoramidite.Masks must be
prefabricated for eachcycleso that the next bases on the synthesized oligonucleotidesare
mapped tothe areas ofexposure. Affymetrix Inc. applied this technology to large-scale
fabrication ofhigh-densityGeneChipprobe arrays. 7
The costs for optics, light sources, and pre-fabricated photolithographic masks can be
prohibitive. This is partly because, in principle,the number ofnecessary masks is four times
the number ofbases inthe oligonucleotides. Texas Instrument’s DMD (DigitalMicromirror
Device) eliminates the necessity for physicalmasks, greatlyreducing processing time
and costs for light-directed oligonucleotide synthesis. A DMD deviceconsists ofan
electromechanically controlled array ofmicromirrors.32It is typicallyused for DLP (digital
light processing)projection displaysystems,with resolutions as high as 2073600 pixels
(SVGA). With flexible programmable light patterns,the generation ofarrays can be
automated.
(R,S)-1-(3,4-(methylenedioxy)-6-nitrophenyl)ethyl chloroformate (MeNPOC) or
2-(2-nitrophenyl)propoxycarbonyl (NPPOC) can be used in photo-deprotection to block
function groups on the linker or monomers.32,33 In one study, a 6003800 DMD array
of 16μm wide micromirrors was used to synthesize 41 mer oligonucleotides.32,33 In this
work, sub-populations of oligonucleotides were eluted, amplified, and assembled.
The authors extrapolated that a chip with 786432 unique 40 mer oligonucleotides could
potentially be used to assemble a .15Mb construct. FlexGen’s in-house synthesizer,
the Flexarrayer, generates oligonucleotides in a similar manner. Nucleotide deprotection
is activated by a laser before the nucleotides are washed and bound to activated spots to
produce 60 mers.
Anotherstudyusedaphotoacidgenerator(PAG),triarylsulfoniumhexafluoroantimonate,
toperformthedeblockingstep.34Aswithconventionalacid-deblockingnucleotide
phosphoramiditechemistry,acidsaregeneratedintheditritylationstep,butwitha
UVphotolyticprocess.TheresultingPGAsolutionisatahighenoughmMconcentration
toeffectivelyremovetheDMTgrouponanucleotide,andismoreefficientthan
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Synthesis and Engineering Tools in Synthetic Biology
photo-cleavagemethods.Themainadvantageofthismethodisthatitrelieson
conventionalphosphoramiditechemistry,withoutthecostoravailabilitylimitations
ofphotolabileprotectiongroups.Photoacidgenerator(PAG)mayliberatefreeradicals
toproduceerrorsinsynthesis.SerafinowskiandGarland35developedandusedtwo
photosensitiveesters,R-phenyl-4,5-dimethoxy-2-nitrobenzyltrichloroacetateandR-phenyl-
4,5-dimethoxy-2,6-dinitrobenzyltrichloroacetate,asPAGagentsforsynthesistoavoid
theseproblems.
Zhou etal.11,36 coupled the PAGtechnique witha parallelized microfluidic platform.
Anarray chip was installed inaflow-through cartridge connected toacommercial
oligonucleotide synthesizer (Expedite 8909). The chips used differentialsurface tension
toisolate reaction sites, andallowed for the miniaturizationof synthesis. Cleaved
oligonucleotides were ofhighenoughquantities and qualitiesto be successfully assembled
in10kbconstructs withPCRandligation.
Electrochemical Arrays
Anothermethod ofDNA synthesis involves using localized electrochemical reactions
for the deblocking step. An array ofplatinum microelectrodes is covered with an electrolyte
solution.Current is simultaneously applied toindividuallyaddressablemicroelectrodes
with asemiconductor circuit,so that the electrolytesnear activated anodes are oxidized and
release acid for deblocking.In one study, the electrolyteusedwas 25mM hydroquinone
and25mM benzoquinone with 25mM tetrabutylammoniumhexafluorophosphate in
anhydrous acetonitrile.37By pairingeachanode withadjacent cathodes which can reduce
acid, the producedacid is confined.The acid thendiffuses tothe layer of substrate which
contains the oligonucleotides beingsynthesized. Theelectrodes were made by thin-film
photolithographyof 50-nm thick iridium metal andweredurableenoughto use over
8 500 cycles without deteriorations. The authors havedemonstrated the synthesis of
short 17bp oligonucleotideswithcompleteelectrochemicaldeblocking in as little as
9 seconds.
Customarray’s oligonucleotides arrays are also synthesized using asemiconductor-based
electrochemical synthesis process. Rather thanusing cathodesto localize generatedacids,
synthesis is performedon apolymersurface over the surfaceof the semiconductor.
Theporous polymerslowsdown acid diffusion toprevent cross-contamination between
local electrodes.Thesurface also increasesthe oligonucleotide density during synthesis.
Customarray’s 90K microarrayscontain 94000 unique oligonucleotides and 25μm
features,andare capable of synthesizing oligonucleotidesof up to 50bp lengths.
Besides featuredensity, another advantageof electrochemicalsynthesis is flexibility.
Becausephotolithographic masks are not involved, it islessexpensive andrequires less
labor to changethe array design. Additionally, electrochemical detritylation is arelatively
efficient chemistry;the deblocking step can be completein secondsand any side reactions
from the electrolyte chemicalsare minimal. Efficient chemistryand alack ofmovingparts
contribute toatotalsynthesis time ofless than 24 hours.
Inkjet Printing
In 1981, Hood and Caruther modifiedliquid phase phosphite-triesterchemistry for solid-
phase DNA synthesis on polymersupports.38Solid-phase synthesis not only became the
method of choicefor conventional DNA synthesis, but also opened DNA synthesis
toautomated and miniaturized strategies on microarrays by eliminatingthe need to purify
synthetic intermediatesor unreacted reagents. The first inkjetDNA synthesizers used
in-house microfabricated piezoelectricactuators.8Piezoelectric inkjet DNA synthesizers
operate similarlytocommercial inkjet printers. Instead of ink,the inkjet head containsfour
phosphoramiditefluidchannels, one activator channel, andone optionalmodified base
CHAPTER 1
New Tools for Cost-Effective DNA Synthesis
DAQ-Electronics
Controller PC
Servo controller
Piezo electric
nozzles
Microchip
FIGURE 1.2
DNAmicrochipsynthesiswithpiezoelectricinkjettechnology.Themicroarraysynthesisplatformconsistsofacontroller
PC,DAQ-electronics,andaservocontroller,whichtogethercontrolthemovementandtheprintingoftheinkjetprinthead
ontoslidesurfaces.Dropletsaredispensedontoasmanyas10000spotfeaturesthatareeachlessthan100μmin
diameter.
channel. In this work, the discrete featureson the substrate couldhold and segregate 100pL
droplets using pre-patterned surface tension (Fig. 1.2).
9
Piezoelectric print head technology is primarily commercialized by Agilent. In 2001,
Hughesetal. used Agilent’ssecond-generation system for gene-expression profilingof
biological samples from various organisms.39 The flexibility ofthe inkjet system allowed
themto create new arrays and update sequencesrapidly. Sincethen,gene expression
profiling has been conventionally performedon Agilent microarrays.39(cid:1)42Through
refinement of surfaces,firing parameters, andsolvent mixtures, Agilent has evolved its
22575 featurethroughput toacurrent 244000 featurethroughput on asingleglass slide.
Theircurrent systemsnow also include computer visualization systems, print head
maintenance modules, and automatedslidehandling.
GENE ASSEMBLY
Chemical oligonucleotide synthesis accumulates errors due to side reactions and
inefficiencies in the stepwise reactions.43 Although oligonucleotides of up to 600bp
in length can be synthesized, yields are extremely low.44 Gene assembly becomes
necessary for synthesis of longer constructs. Almost all current assembly techniques use
a combination of PCR or ligation-based assembly. The advantage of these methods
over restriction digestion/ligation methods is that they can perform scarless and
sequence-independent assembly.
Ligation-basedassemblyusesthermo-stableDNAligasetojoinpre-synthesized
oligonucleotides.Successfuluseofligation-basedassemblyhasbeendemonstratedin
researchandcommercialsynthesisplatforms.45(cid:1)47Thermo-stableligaseisadvantageous
comparedtoT4DNAligase,becausefewerDNAsecondarystructureswillformatelevated
ligationtemperatures.48(cid:1)50Ligation-basedassemblyinvolvesligationandPCRamplification.
Overlappingoligonucleotidesaredesignedtocompletelycoverbothstrandsofthesequence.
Theyarephosphorylatedatthe50-endsfortheligationreaction.Theoligonucleotides
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Synthesis and Engineering Tools in Synthetic Biology
arefirstheatdenatured,andthencooledforannealingandligationat50(cid:1)65(cid:3)C.
Becausetheligationreactionislinear,thefullconstructisdesignedwithflankingprimer
sequencessothatPCRcanamplifytheconstruct.
PCR-based assembly, otherwise knownas polymerase cycling assembly (PCA),remains
one ofthe most cost-effective gene assembly methods.51,52 In atwo-step procedure,
partiallyoverlapping oligonucleotidesare designed tospan 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 sothat overlapping oligonucleotidesannealand extend. The resulting
double-stranded constructcan serveas the template for the PCR amplification reaction. In a
single-stepprocedure,the amplification primersare included with the oligonucleotides for
acombined assembly and amplification reaction.54,55 Although extra cyclesmay be needed,
this procedure is moreeasilymultiplexed than assemblywith multiple steps.53 Although
PCR-based methodsare efficient, constructs involvingrepetitivesequences or secondary
structuresmay havedifficultiesduring PCR, andthus can better be assembled with
ligase-based assembly.56
There are anumber of relatedoverlapping extension techniquesfor sequence-independent
assembly.For the sequence-independent assemblyofcircular double-stranded constructs
or plasmids,circular polymerase extensioncloning(CPEC)57can successfullyassemblenot
only multiple-geneconstructs, but also combinatorial sequencelibraries.19 In asingle-step
reaction,overlapping oligonucleotides are assembled and circularized. Other approaches
suitable for plasmidconstruction include the In-Fusion commercialkit from Clontech,58
Uracil-specific excision reagent(USER)59and Sequence- andLigation-Independent Cloning
(SLIC).60Errorsmayarise duringthe assemblyoflargeconstructs. Gibson isothermal or
‘chewback and anneal’assembly avoids length-dependent errors withthe T5DNA
10 polymerase, allowing for assembly of genome length constructs,61suchas a16.3-kb
mitochondrial genome.62
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.
Althoughhigh-fidelity DNAmicrochipscan hold up toamillion uniqueoligonucleotides,
they are difficult to scale.Microarray oligonucleotide pools are highly complex, andbecome
problematic incomplicationslike potential cross-hybridization between assembled
fragments. More successful scaling will lower the cost of high-quality gene synthesis.
To address this issue, Kosuri etal. combined selectiveoligonucleotide pool amplification,
optimized gene assembly, and enzymatic error correction.12 Theauthors cleaved
microchip-synthesized oligonucleotides. These oligonucleotidesweresynthesized with
flanking amplification primer annealing sites corresponding toseveralsubpools. A quarter-
million specific amplification primers were then used toselectively PCR amplify specific
subpools ofoligonucleotides from the original complex background of oligonucleotides.
Theflanking amplification primer sequenceswerethencleaved with restriction enzymes,
which allows for seamlesssubsequent PCR assembly into gene constructs.Theauthors
tested the system on 47 genes encoding for proteins and antibodies,including 40 error-free
single-chainantibody genesthathad previously been shown tobe difficult tosynthesize
due tohighGCcontent andrepetitive sequences. The assemblyoptimization andenzymatic
treatmentallowed for accurateand low-costsynthesis, bringing costsdown to an estimated
USD0.01/bp.
CHAPTER 1
New Tools for Cost-Effective DNA Synthesis
On-chip amplification and assembly
FIGURE 1.3
Integratedon-chipDNA
amplificationandgene
assembly.Themicrochips
aredividedintophysically
isolatedsub-arrayswhere
oligonucleotidesare
amplifiedbyisothermo
nickingandstrand
Enbolished microchip displacementamplification.
with endented subarrays Thereleasedstrandsare
theassembledinto
0.5(cid:1)1kbgenefragments
withinthewells.
11
QUALITY CONTROL
Eliminatingerrors is critical for most gene synthesis projects, and is probablythe most
costlyandtime-consuming step in gene synthesis. Errors are generated mainly during
chemical oligonucleotide synthesis andalso in subsequentgene assembly steps.
In order toreduce the number offaultyoligonucleotides,chromatographic purification
by high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis
(PAGE)purification is typicallyused by conventional oligonucleotide synthesis vendors.
However, chromatographic purification is expensive andtime-consuming, and PAGE
purification is even more costly. In this section, wewill detailalternative purification
methods that couple withhigh-throughput synthesis technologies.
Fluorescence Selection
Reporter genes can be used to select in-frame gene fusions.Kim et al.used afluorescence
selection method toimprove the fidelity of gene synthesis by focusing on the elimination
of deletionsand insertionsin chemically synthesized genes.Theauthors constructed vectors
where GFP is expressed if the insertion of an in-frame DNA constructinto appropriate
cloning sites shifts the GFP gene in frame.64 Theabsence ofinsertions or deletions results
in fluorescent cell colonies when transformed into E.coli cells, whichcan be selected using
atransilluminator. The authors synthesized sixsimilar-length genesfrom overlapping
oligonucleotidesusingligase-based assemblyandsubsequentPCR, ligated the genes into
pBK reporter vectors,andtransformed for overnight growth of colonies on agar plates.
On average, error rates improved from 1 in 629bp (blind selection)to 1 in 6552bp
(green fluorescenceselection).This error rate allows for the straightforwardconstruction
of error-free genes larger than 1000bp by one-cycle synthesis.
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Synthesis and Engineering Tools in Synthetic Biology
Hybridization Selection
Bringing down error rates of microarray oligonucleotide synthesis would involve
substantial efforts. Microarrays typicallyproduce oligonucleotides with lower product
quality,lower product yields,and higher complexitythan other sources.Tian etal.used
parallel amplification and purification of oligonucleotides from complexoligonucleotide
mixturesusing microfluidics to allow for a2~5-foldincrease in qualityover PAGEpurified
microarray derived oligonucleotides and a10-fold increasein qualityover unpurified
microarray synthesized oligonucleotides.53This was accomplished using generic
amplification primer sequencesfor the amplificationof all oligonucleotides in asingle
PCR reaction. Thelow yields ofmicrochip-derived oligonucleotides are simplyinsufficient
for gene assembly without amplification.Additionally, distinct subsets ofprimer sequences
allow for the fractioning of the oligonucleotide pool into subpools. With aconcentrated,
highly complex oligonucleotide startingpool, the potentialfor undesired cross-
hybridization becomesan issue. Selectiveamplification ofsubsets of oligonucleotides
reduces this cross-hybridization.
Thedifficulty with restriction digesteddouble-stranded microchip oligonucleotides is
that after denaturation, matched antisense oligonucleotides will competewith designated
overlapping oligonucleotides, reducing assembly efficiency. Tian etal. avoided this problem
by enriching single-stranded oligonucleotidesby hybridizationto antisense selection
oligonucleotides and theneluting them. This was also apurification process,which
allowedfor selection against oligonucleotides with errors. These oligonucleotidesproduce
mismatchesduring hybridization andelute at alower temperature. Using selective
amplification and purification via hybridizationenabledthe synthesis of21 genesof the
E.coli 30S ribosomal subunit cluster from asinglepool.
In asimilarapproach, Borovkovet al.combined hybridization-based oligo selection
12
with parallel amplification.14 Erroneousoligos 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 oligonucleotidesfor gene synthesis.
Mismatch Recognition
Besides usinghybridizationselection or improved chemistries with reduced depurination,
ageneral approach for eliminating errors ingene synthesis is touse DNA mismatch
recognition proteins or enzymes.65Mismatchesare smallsingle-strand loops that
are generated by hybridization between correct and incorrect sequences.Thereare two
categories ofenzymes for mismatchremoval: the mismatchbindingproteinsand the
mismatch cleaving enzymes.66(cid:1)70
MismatchbindingproteinslikeMutSselectivelybindtomismatchsites.Incorrectsequences
canbeexcludedbytheremovalofprotein(cid:1)DNAcomplexeswithgel-shiftassayoraffinity
columns.SmithandModrichfirstreportedtheuseofMutHLStoremovemutant
sequences.71MutLcouplesMutHendonucleasetotheMutSboundsite,leadingtoMutH
mediatedcleavage.Thecleavedheteroduplexwasthenremovedbygelelectrophoresis.Using
MutSfromThermusaquaticus,Carretal.establishedamethodthatreducederrorrateto1per
10kbintwocycles.67InordertotoleratemoreerrorsinlongerstartingDNAfragments,MutS
treatmentcanbeappliedtosmallerconstituentfragments.Inaprocesscalledconsensus
shuffling,thelongerfragmentiscleavedintooverlappingfragmentsusingrestriction
endonucleases.Usingcolumnfiltration,immobilizedMutScanbindtoandfiltermismatch
fragments.ElutedfragmentsarethenreassembledwithPCR.Twoiterationsimprovedthe
errorrate3.5-foldto1per3.5kb.68Becauseerroneousgenefragmentsareeliminatedin
mismatchfiltration,enoughinitialfragmentsmustexistinthepooltosurviveMutSbinding.
Thismaypreventmismatchbindingfrombeingappliedtolow-qualityandlow-yield