Amplification and assembly of chip-eluted DNA (AACED): a method for hi
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《核酸研究医学期刊》
1 Center of Nanotechnology, 2 Biotechnology Center, 3 Department of Chemistry, 4 Department of Material Sciences, 5 Department of Electrical and Computer Engineering and 6 Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA
* To whom correspondence should be addressed. Tel: +1 608 262 7712; Fax: +1 608 265 3811; Email: cerrina@nanotech.wisc.edu
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
ABSTRACT
A basic problem in gene synthesis is the acquisition of many short oligonucleotide sequences needed for the assembly of genes. Photolithographic methods for the massively parallel synthesis of high-density oligonucleotide arrays provides a potential source, once appropriate methods have been devised for their elution in forms suitable for enzyme-catalyzed assembly. Here, we describe a method based on the photolithographic synthesis of long (>60mers) single-stranded oligonucleotides, using a modified maskless array synthesizer. Once the covalent bond between the DNA and the glass surface is cleaved, the full-length oligonucleotides are selected and amplified using PCR. After cleavage of flanking primer sites, a population of unique, internal 40mer dsDNA sequences are released and are ready for use in biological applications. Subsequent gene assembly experiments using this DNA pool were performed and were successful in creating longer DNA fragments. This is the first report demonstrating the use of eluted chip oligonucleotides in biological applications such as PCR and assembly PCR.
INTRODUCTION
Recent strides have been made in the de novo synthesis of genes from oligonucleotides, resulting in the assembly of a viral (1) and bacteriophage (2) genome. In both cases, assembly of these long sequences required the use of hundreds of commercially synthesized and gel-purified oligonucleotides. Photolithographic microarray synthesizers are capable of producing hundreds of thousands of unique oligonucleotides quickly and inexpensively, but the elution and utility of such DNA as a template for PCR and assembly PCR has not previously been reported.
In the last decade, the synthesis of DNA on solid substrates has been an important feature of new technologies utilized in genomic studies, particularly for hybridization experiments with microarrays. Since its development, focus has been on advancements in the chemistry (3–5) and efficiency of DNA synthesis (6,7) and engineering improvements that allow for precision patterning (8) and fluorescence analysis (9). Such DNA ‘chips’ also offer the potential for acquiring, de novo, a large number of user-defined DNA oligonucleotide sequences, for subsequent use in biological applications. However, although oligonucleotides grown on slide surfaces have been heavily employed in this manner, knowledge about the amount and relative proportion of failure sequences on the chip surface is lacking.
Previous studies have estimated that a total of 10–30 pmol/cm2 of oligonucleotides are synthesized on the chip surface (6,10). However, it is not clear whether this estimate represents the population of full-length product or a mixture of full-length and truncated or mutated sequences. In studies using photogenerated acids (PGAs) during DNA synthesis (10), it has been postulated that proximity to the synthesis surface led to lower fidelity synthesis, and that this decrease was due to inefficient reactions of various reagents. It is unclear, however, whether such surface effects occur in photolithographic procedures using photolabile 2-nitrophenyl propoxycarbonyl (NPPOC) photodeprotection-based DNA synthesis.
Here, we describe initial studies focusing on the analysis of ssDNA eluted from chips and subsequent optimization for increased amounts of full-length oligonucleotides. A procedure for the amplification and assembly of chip-eluted DNA (‘AACED’) was developed and employed to increase the amount of product to levels and quality more typical of those needed for biological applications. Subsequent tests were then performed to verify the functionality of chip oligonucleotides in applications such as gene assembly.
MATERIALS AND METHODS
DNA synthesis
The modified Maskless Array Synthesizer (MAS) will be referred to as the ‘Biological Exposure and Synthesis System’ or ‘BESS’ (11,12), and the schematic is shown (10) in Figure 1. Each ‘virtual’ mask was created on a computer, and imaged by a Texas Instrument's Digital Light Processor (DLP) which consists of a 1024 x 768 array of 16 μm wide micromirrors. The BESS projected an ultraviolet (UV) image of the virtual mask on the active surface of the glass substrate mounted in a flow-cell reaction chamber connected to a DNA synthesizer. A K?hler illumination system with two dichroic mirrors was used to exclude the infrared and to collect the desired UV light (365 nm) from a 1000 W Hg arc lamp (Oriel Instruments, Stratford, CT). The imaging optics was a 1:1 Offner relay with a 0.08 numerical aperture and a 0.7 spatial partial coherence (13). This instrument has been previously characterized for its ability to produce accurate high-density microarray chips (11,12).
Figure 1. Biological Exposure and Synthesis System (BESS).
The optical system shown in Figure 1 has been optimized with an image-locking subsystem to counter the considerable image drift occurring from thermal expansion of the optical parts within the BESS system (C. Kim, M. Li, M. Rodesch, A. Lowe, K. Richmond, N. Venkaratamaiah and F. Cerrina, submitted for publication). Using a feedback loop, great optical stability with a drift of <0.5 μm over a period of several hours and large temperature variations (C. Kim, M. Li, M. Rodesch, A. Lowe, K. Richmond, N. Venkaratamaiah and F. Cerrina, submitted for publication) has been achieved. The successful application of image locking with an optimized protocol led to a considerable improvement in oligonucleotides gel electrophoresis profiles . For the list of oligonucleotide sequences used see Table 1.
Table 1. Synthesized oligonucleotide sequences
Preparation of synthesis substrates
The slide substrates used for DNA synthesis were either monohydroxysilane slides or slides whose surface was covered with a base-labile linker (T). Monohydroxysilane slides were prepared using Superclean slides from Telechem which were shaken in a solution of 2% N-(3-triethoxysilylpropyl)-4-hydroxybutyramide for 4 h, washed in 95% EtOH for 10 min, and dried in a vacuum oven at 120°C for 1 h. While a base-labile linker (T) has been reported (15), we report here the synthesis of a base-labile linker with a photocleavable protecting group (Figure 2). 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) was obtained from Novabiochem. All other chemicals and solvents were obtained from Aldrich Chemical (Milwaukee, WI). Tetrohydrofuran (THF) was distilled from sodium benzophenone ketyl; dichloromethane from phosphorous pentoxide and pyridine from calcium hydride. Thymidine nucleoside was dried three times over dry pyridine before to use. Analytical thin-layer chromatography (TLC) was carried out on EM Science TLC plates precoated with silica gel 60 F254 (250 μm layer thickness). TLC visualization was accomplished using a UV lamp (250 nm) and/or charring solutions of either basic potassium permanganate or phosphomolybdic acid. Flash column chromatography (FCC) was performed on Silicycle silica gel 60 (230–400 mesh). Proton nuclear magnetic resonance (1H NMR) spectra were recorded in deuterated solvents on a Bruker AC-300 (300 MHz). Chemical shifts are reported in parts per million ( relative to tetramethylsilane (TMS, 0.00) or relative to residual solvent signal (CDCl3 7.24). Coupling constants (J-values) are given in Hz, and peak multiplicities are denoted by s (singlet), d (doublet), dd (doublet of doublets), m (multiplet), q (quartet). High-resolution electrospray ionization mass spectra were obtained on a Micromass LCT.
Figure 2. Chemical synthesis of a novel base labile linker.
Synthesis of 1 A solution of 2-(2-nitrophenyl)-propan-1-ol (16,17) (2.70 g, 14.9 mmol) in dry THF (100 ml) was added dropwise to an ice-cooled 20% solution of phosgene in toluene (8 eq., 60 ml) over 30 min. The mixture was stirred at 0°C for 30 min and for a further 2 h at room temperature. The reaction was monitored until completion by TLC and the solvents removed in vacuo through a sodium hydroxide trap. The desired chloroformate intermediate was obtained in 80% yield (2.91 g) and taken up immediately in dry methylene chloride (100 ml) and added dropwise to a cooled solution (ice/methanol bath, –15°C) of dry thymidine (1.2 eq., 3.5 g) over 45 min. The reaction was allowed to come to room temperature overnight (12 h) before the solvents were removed in vacuo. FCC provided the desired product 1 (3.44 g, 64%). 1H NMR (300 MHz, CDCl3) 8.88 (bs, 1H, NH), 7.50–7.40 (d, J = 1 Hz, m, 5H, 4 aromatic Hs, vinylic H), 6.19 (m, 1H, O(N)CHCH2), 5.23 (d, J = 5.7 Hz, 1H, CH2CHCHOH), 4.44–4.28 (m, 2H, CHCH2OCO), 4.16 (d, J = 7 Hz, 1 H, CH2CHCHOH), 4.00–3.85 (m, 2H, CH2CHCHOH), 3.87 (q, 1H, benzylic H), 2.51–2.35 (m, 1H, O(N)CHCH2), 1.95 (s, 3H, = CCH3), 1.41 (d, J = 6.9 Hz). Exact mass measurement + calculated: 472.1332, measured: 472.1323.
Synthesis of 2. Succinic anhydride (1 eq. 142 mg) was added in portions over 30 min to a stirred solution of 1 (1 eq. 1.4 mmol. 640 mg) dissolved in dry pyridine (15 ml) containing P-dimethylaminopyride (DMAP) (0.5 eq., 87 mg). The reaction was stirred overnight and the solvents removed in vacuo. Flash column chromatography provided base-cleavable thymidine linker 2 (0.74 g, 95%). 1H NMR (300 MHz, CDCl3) 9.87 (bs, 1H, NH), 7.73 (d, J = 8.1 Hz, 1H, aromatic H), 7.59–7.30 (m, 4H, 3 aromatic Hs, vinylic H), 6.31 (m, 1H, O(N)CHCH2), 5.28–5.19 (dd, J = 6.6 and 6.3 Hz, 1H, O(N)CHCH2CHO), 4.43–4.19 (m, 5H, overlapping CH2CH(O)CH(O), CHCH2OCO and CH2CH(O)CH(O)), 4.16 (d, J = 7 Hz, 1H, CH2CHCHOH), 4.00–3.85 (m, 2H, CH2CHCHOH), 3.77 (q, 1H, benzylic H), 2.66 (d, 4H, OC(O)CH2CH2CO2H), 2.43 (m, 1H, O(N)CHCHH?), 2.16 (m, 1H, O(N)CHCHH?), 1.79 (d, 3H, vinylic CH3), 1.35 (d, J = 7.1 Hz). Exact mass measurement + calculated: 548.1517, measured: 548.1490.
Attachment of linker 2 onto glass slides
Base-cleavable linker 2 (7 mg, 14 μmol), HBTU (1 eq., 6 mg), DMAP (1 eq., 2 mg) and dry di-isopropyethylamine (10 μl) in dry acetonitrile (300 μl) were mixed in a sealed vial and immediately transferred to dry amine-derivatized glass slides (SuperAmine Substrates from Telechem). Fifty microliters of the reaction solution was incubated in each slide under a cover slip for 20 min in the dark. The slide was washed with copious quantities of acetonitrile and dried with a stream of nitrogen before being stored in a dessicator before use.
DNA synthesis reagents were purchased from Proligo (Boulder, CO), Glen Research (Sterling, VA) and Nimblegen Systems (Madison, WI). The DNA synthesis process underwent optimization , with changes occurring in the length of coupling times, UV exposure times and the introduction of an image locking system. Hybridization of chips was performed as noted (12) except that the 2 h hybridizations were performed with Cy3-labeled probes, with subsequent scanning and analysis performed on an Applied Precision ArrayWorx Biochip Reader.
Elution of oligonucleotides
Oligonucleotides used for gel analysis were cleaved from either base-labile or monohydroxysilane slides by incubation with ethylenediamine:ethanol (1:1) at room temperature for 3 h, or with NH4OH for 1 h, respectively. The eluate was dried down in a speed vacuum centrifuge (240 g) and the precipitate resuspended in 5 μl sterile Milli-Q water. Oligonucleotides used in biological applications were synthesized either on slides with a base-labile linker (T) or on monohydroxysilane slides, and were cleaved from the slide surface by incubating with NH4OH at room temperature for 30–60 min, respectively. The cleaved oligonucleotides were then incubated in NH4OH for an additional 16 h to remove base-protecting groups. The eluate was dried down and resuspended as noted above.
Gel analysis
Radiolabeling of chip eluates and some assembly products was performed by labeling DNA with -ATP (Amersham #AA0068; 3000 Ci/mmol; 10 μCi/μl) using T4 polynucleotide kinase (Promega) and analyzed by gel electrophoresis on a 1x TBE–20% polyacrylamide–7 M urea gel with 1x TBE running buffer. (Gels utilized 39 x 20 cm2 plates using a 0.8 mm spacer.) After electrophoresis at 1500 V for 1 h, the gel was placed in a phosphoimager cassette and scanned using the STORM Molecular Dynamics system. Analysis of the 180 bp assembly product was performed on a 3.5% TBE–agarose gel, electrophoresized at 100 V for 45 min, then stained with ethidium bromide and visualized by UV illumination.
MALDI-TOF MS instrument and sample conditions
All MALDI-TOF MS was performed in linear negative ion mode ona Bruker Biflex III. Desorption/ionization was performed with a 337 nm N2 laser. In each case, an accelerating voltage of 19 kV was used along with a 150 ns delayed extraction. Data were collected at 500 Mhz with a Lecroy LSA-1000 digitizer. A co-matrix of nicontinic acid and anthranilic acid (Sigma-Aldrich) was used (12.3:27.4 mg) in acetronitrile and H2O (500:300 ml). Oligonucleotides were desalted with C18 ZipTips (Millipore) prior to spotting onto a Teflon-coated Anchor plate (Bruker) and were mixed with the co-matrix before drying (18).
‘AACED’
A portion of the eluted oligonucleotide were used for amplification with two 15 base PCR primers containing the restriction enzyme site (MlyI; see Table 1) and Pfu polymerase (Stratagene). After amplification the product of the reaction was digested with MlyI at 37°C. Directly eluted oligonucleotides or MlyI-digested amplified oligonucleotides were used as the basis for gene-assembly reactions. Assembly reactions were performed in a ‘primerless’ PCR with Pfu polymerase, deoxyribonucleotides and buffer, for 25 cycles . An aliquot of the assembly reaction was then used in an amplification reaction with flanking primers, Pfu polymerase, deoxyribonucleotides and buffer, and run for 35 cycles . Diagnostic digests were performed for 1 h at 37°C.
RESULTS
Chip production
To determine whether ssDNA eluted from microarray chips was suitable for use in biological applications, initial studies were performed to characterize the oligonucleotides and optimize the synthesis of full-length products. To obtain a flexible and rapid source of oligonucleotides, a Biological Exposure and Synthesis System (BESS) (Figure 1) was used to synthesize DNA on glass slides. The BESS system is capable of synthesizing up to 786 432 oligonucleotide species in discrete 16 μm2 regions on the surface of glass slides (11). These instruments have been previously characterized for their ability to produce accurate high-density microarray chips (11,12) through the use of digital light processors (DLPs; Texas Instruments) and photolabile 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC) chemistry (3,4). In these experiments, a chip area of (1.7 x 1.3 cm2) was used for DNA synthesis.
Oligonucleotide analysis
To examine the synthesized DNA profiles and optimize conditions for synthesis of the desired full-length product, monohydroxysilane chips were synthesized with homopolymers (T10) using either short (20 s) or long (6 h) coupling of the first base. Lengthening of the first coupling time was one parameter considered potentially important since it maximizes the coupling to available reactive sites on the glass surface. The DNA was then eluted and analyzed by gel electrophoresis of the short (Figure 3, lane 1) and long (Figure 3, lane 2) T10 synthesis products. This result was confirmed by mass spectroscopy (18) (data not shown). Profiles of the products show an 80% increase in the amount of T10 product. Therefore, it is key to note that by alteration of only one parameter, the quantity of full-length DNA synthesized can be dramatically affected.
Figure 3. Oligonucleotide analysis short and long T10 coupling products were eluted from monohydroxysilane slides, labeled with ATP and analyzed. Radiolabeled short (lane 1) and long (lane 2) coupling products were electrophoresed on a 20% PAGE–7 M urea 1x TBE gel.
The synthesis products of chips with homopolymers (T12) were also compared to that of heteropolymers (M12) (Figure 4; for sequence, see Table 1). Gel analysis of the synthesized M12 (mixed base) oligonucleotides (Figure 4a, lane 2) revealed a dramatic decrease in the quantity of full-length product as compared to the amount of full-length product of T12 (Figure 4a, lane 1) and these profile differences were noted regardless of the type of chip substrate used. In an attempt to increase the amounts of full-length DNA synthesized, additional modifications were performed altering the parameters of coupling time (3-fold extension), exposure time (3-fold increase), and through the addition of protocol steps (i.e. Ar drying step) . Subsequent optimization of AGS parameters allowed for a greater proportion of full-length oligonucleotide production, as noted here during the production of (2) 41 base heteropolymers (M41a, M41b) (Figure 4b). The gel profile of the synthesis products from the ‘unoptimized’ chip (Figure 4b, lane 1) showed little to no evidence of a 41 base oligonucleotide. However, gel profile of the products from the ‘optimized’ chip (Figure 4b, lane 2) displayed a significant band of the correct size. Quantitation of the M41 gel profiles (Figure 4b, lanes 1 and 2), revealed a 100-fold increase in the amount of full-length product.
Figure 4. Homo- and Heteropolymer synthesis and optimization. All products were eluted from base-labile linker slides, electrophoresed on a 20% polyacrylamide–7 M urea 1x TBE gel . (a) Homopolymers (T12; lane 1) and heteropolymers (M12; lane 2). (b) Heteropolymers (M41a, M41b) pre-optimization (M41a, M41b) (lane 1) and post-optimization (M41a, M41b) (lane 2).
Biological application
To confirm identity and determine whether the eluted oligonucleotides were biologically functional, a limited gene assembly (Figure 5) was performed using non-amplified ssDNA oligonucleotides eluted directly from chip surface or amplified dsDNA (‘AACED’) oligonucleotides. For non-amplified oligonucleotides to be used for extension during gene assembly, it was necessary that their 3' ends retain a hydroxyl-group (–OH). To accomplish this, oligonucleotides were synthesized on chips containing a base-labile linker which, upon treatment with base (NH4OH), releases oligonucleotides with 3'-OH groups.
Figure 5. Elution and assembly of chip oligonucleotides. Oligonucleotides were obtained by (a) synthesis on base-labile or monohydroxysilane slides after cleavage and post-processing steps. (b) Assembly occurred with eluted oligonucleotides which had been designed to contain a region of overlap to allow for annealing and extension.
To ‘assemble’ DNA sequences, overlapping sense and reverse complementary oligonucleotides were designed (Table 1), and, through assembly and amplification protocols, a longer DNA sequence was formed. This method was chosen due to previous work from our laboratory which has shown that the products of various gene assembly protocols have inherent differences in error rates. In our hands the PCR assembly reactions were the most successful. The procedures relying on PCR yielded products with lower error rates (K. E. Richmond, manuscript in preparation) and therefore was the assembly method used in this work. The identity of the amplified products derived from the assembly of eluted oligonucleotides was confirmed through the addition of diagnostic restriction enzyme sites to the designed sequence. The foundation for oligonucleotide design was based on the sequence of Green Fluorescent Protein gene (GFPuv, Clontech).
‘Non-amplified’ oligonucleotides (chip eluates) were tested for their ability to assemble. Two (41 base) oligonucleotides (M41c, M41d) were synthesized and subsequently released from base-labile linkers for use in a minimal gene assembly process to form a 61 base fragment (Figure 6a, lane 1). The identity of the amplified assembled fragment was confirmed with a diagnostic digest (Figure 6a, lane 2) and analyzed on a denaturing polyacrylamide gel.
Figure 6. Application of chip oligonucleotides. (a) assembly of (2) eluted 41 base oligonucleotides; Radiolabeled assembly product digested with EcoRI (lane 1) and undigested (lane 2). Std: Oligo Ladder (Amersham) 8–32 bases; 60: a 60 base oligonucleotides standard. (b) Radiolabeled product of ‘Booster’ amplification (lane 1); product(lane 1) digested with PvuII (lane 2); product(lane 1) digested with MlyI (lane 3). Analysis of (a) and (b) 32P-labeled products on a 20% polyacrylamide–7 M urea 1x TBE gel. (c) Assembly of ‘AACED’ amplified product (lane 1); product (lane 1) digested with XhoI (lane 2). Std: 100 bp ladder (Promega).
We proposed that a possible drawback for using chip-eluted ssDNA as the direct input for some biological applications could be the inherently small concentrations available for use. To dramatically increase the overall amount of chip DNA, an amplification strategy called ‘amplification of assembled chip-eluted DNA’ was tested. AACED allows for the selection and amplification of full-length chip oligonucleotide populations, followed by the subsequent, enzyme-mediated removal of primer sequences flanking the unique internal DNA sequences (Table 1). The ability to obtain a diverse, large set of oligonucleotides in this manner may revolutionalize many biological applications since it provides access to the hundreds of thousands of user-defined sequences created on glass surfaces. In our experiments, the restriction enzyme MlyI was designed into upstream and downstream primer sequences to allow for complete blunt removal of primer sequences post-amplification. Therefore, after producing high copy numbers of the full oligonucleotides, the flanking primer sequences are removed, yielding the variable internal oligonucleotide sequences ready for use in any number of applications.
As a proof of concept, a single 60 base oligonucleotide was synthesized, eluted, amplified (Figure 6b, lane1) and digested with MlyI (Figure 6b, lane 3) to allow for the release of the upstream and downstream flanking 15 base primer sequences, leaving a single 30 base oligonucleotide for subsequent applications. The digested sample was then labeled using ATP and T4 polynucleotide kinase and analyzed on a denaturing polyacrylamide gel. The amplified product from the chip eluate (Figure 6b, lane 1) gave a strong single band of the correct size (60 bp) which digested as expected with the diagnostic restriction enzyme (Figure 6b, lane 2). To determine whether the MlyI segments were efficiently released, the digested sample was also radiolabeled and analyzed (Figure 6b, lane 3) and the only band evident was a doublet of the correct size (15 bp). This doublet is postulated to occur due to the slight differences in molecular weight between the released, mixed-base 15 base fragments. Due to the presence of 5' phosphates, the internal 30 base fragments were not labeled and therefore not visible in the lane containing the Mly1 digestion (Figure 6b, lane 3). Of note is the lack of the initial 60 base band (Figure 6b, lane 3), indicating that all the input DNA was thoroughly digested with MlyI.
With the ‘AACED’ concept verified, larger assemblies of eight overlapping sense and reverse complement oligonucleotides were performed (Figure 6c). The synthesized oligonucleotides (Table 1) were 70 bases in length, with the initial and final 15 bases designed for primer sequences, leaving an internal 40 base sequence for subsequent use in assembly. Analysis of the amplified assembly product revealed a band of the expected assembled product size (180 bp) (Figure 6c, lane 1) and its identity was confirmed by a diagnostic digest (Figure 6c, lane 2).
DISCUSSION
Prior to performing this analysis of chip-eluted oligonucleotides, modifications of the maskless array synthesizer were performed to allow for high-quality, high-density synthesis of long oligonucleotides onto glass slides. The enhancements utilized existing technology such as an image-locking system to allow for the improved, accurate synthesis of long oligonucleotides up to 90 bases in length, as shown in imaging and fluorescence studies . This technique allows for the synthesis of not only longer oligonucleotides, but also more accurate oligonucleotides (by reducing inaccurate deprotection which causes subsequent base addition and/or deletion), and the ability to create more sequences per chip (reducing the need for ‘border’ areas to limit crossover deprotection of adjoining regions from drift).
One significant finding was the dramatic effect noted by the alteration of a single synthesis parameter on the quantity of full-length oligonucleotides produced. It is evident that the ‘standard’ method for DNA synthesis, although adequate for microarray production and use, may not be the best manner in which to synthesize large quantities of full-length oligonucleotides. Analysis of the synthesis products of the homopolymer T10 (Figure 3, lane 2) revealed that only a minority of oligonucleotides were full length (<35% of population). This was interesting since the coupling efficiency for NPPOC-T had been reported previously (98%) and for a T10, therefore, 76% of products were expected to be full length. Such differences between the theoretical and experimental full-length oligonucleotides production could be accounted for by a myriad of reasons such as inefficiencies in deprotection (4), coupling, or cleavage from the chip surface. Prior to this work, no analysis of chip eluates had been done on chips generated with NPPOC chemistry. These results led us to optimize the DNA synthesis parameters (coupling, exposure time, protocol steps) used in this study.
The difference in profiles between mixed base and poly(T) oligonucleotides (Figure 4a) was significant and intriguing. Although the efficiencies of base addition have been reported previously (11) and shown to vary for the different NPPOC phosphoramidites (A-96%, G-97%, T-98% and C-99%), it is unlikely that these differences alone account for the decrease in M12 full-length products. Further optimization studies were performed to increase the amount of full-length product and it was noted that a 100-fold increase occurred not by changing the basic chemistry or reagents used, but through changes in the synthesis protocol itself. The combination of increased coupling time (from 20 to 60 s), increased exposure time(from 50 to 150 s), the addition of Ar drying steps, and an image-locking system was used to produce more full-length product even for longer oligonucleotides such as 40mers (Figure 4b).
Interestingly, chips that were not optimized for DNA synthesis yielded strong hybridization profiles (data not shown). Despite the lack of full-length oligonucleotides seen in gel profiles, hybridization appears sensitive enough to detect these species, present in only small percentages, and, therefore, hybridization results do not accurately determine overall oligonucleotide quality; they are merely indicative of trends in purity. In high-density arrays there are also additional factors which can affect the interpretation of hybridization results and make these results unreliable for any predicative use for chip oligonucleotide quality.
Initial experiments with oligonucleotides (41mers) from ‘optimized’ syntheses on base-labile slides were successful in assembling to form a larger DNA fragment (61mers). This is the first reported use of the base-labile linkage for release of DNA from chips and the use of eluted chip DNA in a biological application. Assemblies using amplified and digested oligonucleotides (eight 70mers digested to yield eight 40mers) were also successful and this method could be applied to create large populations of oligonucleotides for other biological purposes. The ‘AACED’ amplifying method has the advantage of not only increasing the number of oligonucleotides for use, but it also allows for the enrichment of the full-length product over truncate species. Additionally this method could allow for amplification of sub-populations of DNA from the chip surface, making each chip useful for a variety of oligonucleotides syntheses. Indeed, with the (overly) optimistic assumption of obtaining 10 pmol/cm2 of oligonucleotides from a chip, this quantity would correspond to 12 attomol/pixel and thus without amplification, most biological applications would not be feasible. If our AACED was extended to its full potential, a single chip with 786 432 unique 40mer oligonucleotides could potentially be used to assemble >15 Mb of DNA.
Miniaturization of hardware provides a means to reduce the quantities of reagents and therefore the costs of oligonucleotide synthesis. Technological applications using oligonucleotides, however, are becoming very diverse and hence the variety of sequences needed is escalating. In keeping with this trend, we have utilized a Biological Exposure Synthesis System (BESS) to produce chips with small amounts of synthesized oligonucleotides for subsequent elution and utilization in biological assays. This system can allow for hundreds of thousands of discrete oligonucleotides species to be made inexpensively and quickly for utilization in a myriad of biological assays, including that of gene assembly.
ACKNOWLEDGEMENTS
We would like to acknowledge that this work was supported by DARPA Grant No. DAAD 19-02-2-0026.
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* To whom correspondence should be addressed. Tel: +1 608 262 7712; Fax: +1 608 265 3811; Email: cerrina@nanotech.wisc.edu
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
ABSTRACT
A basic problem in gene synthesis is the acquisition of many short oligonucleotide sequences needed for the assembly of genes. Photolithographic methods for the massively parallel synthesis of high-density oligonucleotide arrays provides a potential source, once appropriate methods have been devised for their elution in forms suitable for enzyme-catalyzed assembly. Here, we describe a method based on the photolithographic synthesis of long (>60mers) single-stranded oligonucleotides, using a modified maskless array synthesizer. Once the covalent bond between the DNA and the glass surface is cleaved, the full-length oligonucleotides are selected and amplified using PCR. After cleavage of flanking primer sites, a population of unique, internal 40mer dsDNA sequences are released and are ready for use in biological applications. Subsequent gene assembly experiments using this DNA pool were performed and were successful in creating longer DNA fragments. This is the first report demonstrating the use of eluted chip oligonucleotides in biological applications such as PCR and assembly PCR.
INTRODUCTION
Recent strides have been made in the de novo synthesis of genes from oligonucleotides, resulting in the assembly of a viral (1) and bacteriophage (2) genome. In both cases, assembly of these long sequences required the use of hundreds of commercially synthesized and gel-purified oligonucleotides. Photolithographic microarray synthesizers are capable of producing hundreds of thousands of unique oligonucleotides quickly and inexpensively, but the elution and utility of such DNA as a template for PCR and assembly PCR has not previously been reported.
In the last decade, the synthesis of DNA on solid substrates has been an important feature of new technologies utilized in genomic studies, particularly for hybridization experiments with microarrays. Since its development, focus has been on advancements in the chemistry (3–5) and efficiency of DNA synthesis (6,7) and engineering improvements that allow for precision patterning (8) and fluorescence analysis (9). Such DNA ‘chips’ also offer the potential for acquiring, de novo, a large number of user-defined DNA oligonucleotide sequences, for subsequent use in biological applications. However, although oligonucleotides grown on slide surfaces have been heavily employed in this manner, knowledge about the amount and relative proportion of failure sequences on the chip surface is lacking.
Previous studies have estimated that a total of 10–30 pmol/cm2 of oligonucleotides are synthesized on the chip surface (6,10). However, it is not clear whether this estimate represents the population of full-length product or a mixture of full-length and truncated or mutated sequences. In studies using photogenerated acids (PGAs) during DNA synthesis (10), it has been postulated that proximity to the synthesis surface led to lower fidelity synthesis, and that this decrease was due to inefficient reactions of various reagents. It is unclear, however, whether such surface effects occur in photolithographic procedures using photolabile 2-nitrophenyl propoxycarbonyl (NPPOC) photodeprotection-based DNA synthesis.
Here, we describe initial studies focusing on the analysis of ssDNA eluted from chips and subsequent optimization for increased amounts of full-length oligonucleotides. A procedure for the amplification and assembly of chip-eluted DNA (‘AACED’) was developed and employed to increase the amount of product to levels and quality more typical of those needed for biological applications. Subsequent tests were then performed to verify the functionality of chip oligonucleotides in applications such as gene assembly.
MATERIALS AND METHODS
DNA synthesis
The modified Maskless Array Synthesizer (MAS) will be referred to as the ‘Biological Exposure and Synthesis System’ or ‘BESS’ (11,12), and the schematic is shown (10) in Figure 1. Each ‘virtual’ mask was created on a computer, and imaged by a Texas Instrument's Digital Light Processor (DLP) which consists of a 1024 x 768 array of 16 μm wide micromirrors. The BESS projected an ultraviolet (UV) image of the virtual mask on the active surface of the glass substrate mounted in a flow-cell reaction chamber connected to a DNA synthesizer. A K?hler illumination system with two dichroic mirrors was used to exclude the infrared and to collect the desired UV light (365 nm) from a 1000 W Hg arc lamp (Oriel Instruments, Stratford, CT). The imaging optics was a 1:1 Offner relay with a 0.08 numerical aperture and a 0.7 spatial partial coherence (13). This instrument has been previously characterized for its ability to produce accurate high-density microarray chips (11,12).
Figure 1. Biological Exposure and Synthesis System (BESS).
The optical system shown in Figure 1 has been optimized with an image-locking subsystem to counter the considerable image drift occurring from thermal expansion of the optical parts within the BESS system (C. Kim, M. Li, M. Rodesch, A. Lowe, K. Richmond, N. Venkaratamaiah and F. Cerrina, submitted for publication). Using a feedback loop, great optical stability with a drift of <0.5 μm over a period of several hours and large temperature variations (C. Kim, M. Li, M. Rodesch, A. Lowe, K. Richmond, N. Venkaratamaiah and F. Cerrina, submitted for publication) has been achieved. The successful application of image locking with an optimized protocol led to a considerable improvement in oligonucleotides gel electrophoresis profiles . For the list of oligonucleotide sequences used see Table 1.
Table 1. Synthesized oligonucleotide sequences
Preparation of synthesis substrates
The slide substrates used for DNA synthesis were either monohydroxysilane slides or slides whose surface was covered with a base-labile linker (T). Monohydroxysilane slides were prepared using Superclean slides from Telechem which were shaken in a solution of 2% N-(3-triethoxysilylpropyl)-4-hydroxybutyramide for 4 h, washed in 95% EtOH for 10 min, and dried in a vacuum oven at 120°C for 1 h. While a base-labile linker (T) has been reported (15), we report here the synthesis of a base-labile linker with a photocleavable protecting group (Figure 2). 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) was obtained from Novabiochem. All other chemicals and solvents were obtained from Aldrich Chemical (Milwaukee, WI). Tetrohydrofuran (THF) was distilled from sodium benzophenone ketyl; dichloromethane from phosphorous pentoxide and pyridine from calcium hydride. Thymidine nucleoside was dried three times over dry pyridine before to use. Analytical thin-layer chromatography (TLC) was carried out on EM Science TLC plates precoated with silica gel 60 F254 (250 μm layer thickness). TLC visualization was accomplished using a UV lamp (250 nm) and/or charring solutions of either basic potassium permanganate or phosphomolybdic acid. Flash column chromatography (FCC) was performed on Silicycle silica gel 60 (230–400 mesh). Proton nuclear magnetic resonance (1H NMR) spectra were recorded in deuterated solvents on a Bruker AC-300 (300 MHz). Chemical shifts are reported in parts per million ( relative to tetramethylsilane (TMS, 0.00) or relative to residual solvent signal (CDCl3 7.24). Coupling constants (J-values) are given in Hz, and peak multiplicities are denoted by s (singlet), d (doublet), dd (doublet of doublets), m (multiplet), q (quartet). High-resolution electrospray ionization mass spectra were obtained on a Micromass LCT.
Figure 2. Chemical synthesis of a novel base labile linker.
Synthesis of 1 A solution of 2-(2-nitrophenyl)-propan-1-ol (16,17) (2.70 g, 14.9 mmol) in dry THF (100 ml) was added dropwise to an ice-cooled 20% solution of phosgene in toluene (8 eq., 60 ml) over 30 min. The mixture was stirred at 0°C for 30 min and for a further 2 h at room temperature. The reaction was monitored until completion by TLC and the solvents removed in vacuo through a sodium hydroxide trap. The desired chloroformate intermediate was obtained in 80% yield (2.91 g) and taken up immediately in dry methylene chloride (100 ml) and added dropwise to a cooled solution (ice/methanol bath, –15°C) of dry thymidine (1.2 eq., 3.5 g) over 45 min. The reaction was allowed to come to room temperature overnight (12 h) before the solvents were removed in vacuo. FCC provided the desired product 1 (3.44 g, 64%). 1H NMR (300 MHz, CDCl3) 8.88 (bs, 1H, NH), 7.50–7.40 (d, J = 1 Hz, m, 5H, 4 aromatic Hs, vinylic H), 6.19 (m, 1H, O(N)CHCH2), 5.23 (d, J = 5.7 Hz, 1H, CH2CHCHOH), 4.44–4.28 (m, 2H, CHCH2OCO), 4.16 (d, J = 7 Hz, 1 H, CH2CHCHOH), 4.00–3.85 (m, 2H, CH2CHCHOH), 3.87 (q, 1H, benzylic H), 2.51–2.35 (m, 1H, O(N)CHCH2), 1.95 (s, 3H, = CCH3), 1.41 (d, J = 6.9 Hz). Exact mass measurement + calculated: 472.1332, measured: 472.1323.
Synthesis of 2. Succinic anhydride (1 eq. 142 mg) was added in portions over 30 min to a stirred solution of 1 (1 eq. 1.4 mmol. 640 mg) dissolved in dry pyridine (15 ml) containing P-dimethylaminopyride (DMAP) (0.5 eq., 87 mg). The reaction was stirred overnight and the solvents removed in vacuo. Flash column chromatography provided base-cleavable thymidine linker 2 (0.74 g, 95%). 1H NMR (300 MHz, CDCl3) 9.87 (bs, 1H, NH), 7.73 (d, J = 8.1 Hz, 1H, aromatic H), 7.59–7.30 (m, 4H, 3 aromatic Hs, vinylic H), 6.31 (m, 1H, O(N)CHCH2), 5.28–5.19 (dd, J = 6.6 and 6.3 Hz, 1H, O(N)CHCH2CHO), 4.43–4.19 (m, 5H, overlapping CH2CH(O)CH(O), CHCH2OCO and CH2CH(O)CH(O)), 4.16 (d, J = 7 Hz, 1H, CH2CHCHOH), 4.00–3.85 (m, 2H, CH2CHCHOH), 3.77 (q, 1H, benzylic H), 2.66 (d, 4H, OC(O)CH2CH2CO2H), 2.43 (m, 1H, O(N)CHCHH?), 2.16 (m, 1H, O(N)CHCHH?), 1.79 (d, 3H, vinylic CH3), 1.35 (d, J = 7.1 Hz). Exact mass measurement + calculated: 548.1517, measured: 548.1490.
Attachment of linker 2 onto glass slides
Base-cleavable linker 2 (7 mg, 14 μmol), HBTU (1 eq., 6 mg), DMAP (1 eq., 2 mg) and dry di-isopropyethylamine (10 μl) in dry acetonitrile (300 μl) were mixed in a sealed vial and immediately transferred to dry amine-derivatized glass slides (SuperAmine Substrates from Telechem). Fifty microliters of the reaction solution was incubated in each slide under a cover slip for 20 min in the dark. The slide was washed with copious quantities of acetonitrile and dried with a stream of nitrogen before being stored in a dessicator before use.
DNA synthesis reagents were purchased from Proligo (Boulder, CO), Glen Research (Sterling, VA) and Nimblegen Systems (Madison, WI). The DNA synthesis process underwent optimization , with changes occurring in the length of coupling times, UV exposure times and the introduction of an image locking system. Hybridization of chips was performed as noted (12) except that the 2 h hybridizations were performed with Cy3-labeled probes, with subsequent scanning and analysis performed on an Applied Precision ArrayWorx Biochip Reader.
Elution of oligonucleotides
Oligonucleotides used for gel analysis were cleaved from either base-labile or monohydroxysilane slides by incubation with ethylenediamine:ethanol (1:1) at room temperature for 3 h, or with NH4OH for 1 h, respectively. The eluate was dried down in a speed vacuum centrifuge (240 g) and the precipitate resuspended in 5 μl sterile Milli-Q water. Oligonucleotides used in biological applications were synthesized either on slides with a base-labile linker (T) or on monohydroxysilane slides, and were cleaved from the slide surface by incubating with NH4OH at room temperature for 30–60 min, respectively. The cleaved oligonucleotides were then incubated in NH4OH for an additional 16 h to remove base-protecting groups. The eluate was dried down and resuspended as noted above.
Gel analysis
Radiolabeling of chip eluates and some assembly products was performed by labeling DNA with -ATP (Amersham #AA0068; 3000 Ci/mmol; 10 μCi/μl) using T4 polynucleotide kinase (Promega) and analyzed by gel electrophoresis on a 1x TBE–20% polyacrylamide–7 M urea gel with 1x TBE running buffer. (Gels utilized 39 x 20 cm2 plates using a 0.8 mm spacer.) After electrophoresis at 1500 V for 1 h, the gel was placed in a phosphoimager cassette and scanned using the STORM Molecular Dynamics system. Analysis of the 180 bp assembly product was performed on a 3.5% TBE–agarose gel, electrophoresized at 100 V for 45 min, then stained with ethidium bromide and visualized by UV illumination.
MALDI-TOF MS instrument and sample conditions
All MALDI-TOF MS was performed in linear negative ion mode ona Bruker Biflex III. Desorption/ionization was performed with a 337 nm N2 laser. In each case, an accelerating voltage of 19 kV was used along with a 150 ns delayed extraction. Data were collected at 500 Mhz with a Lecroy LSA-1000 digitizer. A co-matrix of nicontinic acid and anthranilic acid (Sigma-Aldrich) was used (12.3:27.4 mg) in acetronitrile and H2O (500:300 ml). Oligonucleotides were desalted with C18 ZipTips (Millipore) prior to spotting onto a Teflon-coated Anchor plate (Bruker) and were mixed with the co-matrix before drying (18).
‘AACED’
A portion of the eluted oligonucleotide were used for amplification with two 15 base PCR primers containing the restriction enzyme site (MlyI; see Table 1) and Pfu polymerase (Stratagene). After amplification the product of the reaction was digested with MlyI at 37°C. Directly eluted oligonucleotides or MlyI-digested amplified oligonucleotides were used as the basis for gene-assembly reactions. Assembly reactions were performed in a ‘primerless’ PCR with Pfu polymerase, deoxyribonucleotides and buffer, for 25 cycles . An aliquot of the assembly reaction was then used in an amplification reaction with flanking primers, Pfu polymerase, deoxyribonucleotides and buffer, and run for 35 cycles . Diagnostic digests were performed for 1 h at 37°C.
RESULTS
Chip production
To determine whether ssDNA eluted from microarray chips was suitable for use in biological applications, initial studies were performed to characterize the oligonucleotides and optimize the synthesis of full-length products. To obtain a flexible and rapid source of oligonucleotides, a Biological Exposure and Synthesis System (BESS) (Figure 1) was used to synthesize DNA on glass slides. The BESS system is capable of synthesizing up to 786 432 oligonucleotide species in discrete 16 μm2 regions on the surface of glass slides (11). These instruments have been previously characterized for their ability to produce accurate high-density microarray chips (11,12) through the use of digital light processors (DLPs; Texas Instruments) and photolabile 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC) chemistry (3,4). In these experiments, a chip area of (1.7 x 1.3 cm2) was used for DNA synthesis.
Oligonucleotide analysis
To examine the synthesized DNA profiles and optimize conditions for synthesis of the desired full-length product, monohydroxysilane chips were synthesized with homopolymers (T10) using either short (20 s) or long (6 h) coupling of the first base. Lengthening of the first coupling time was one parameter considered potentially important since it maximizes the coupling to available reactive sites on the glass surface. The DNA was then eluted and analyzed by gel electrophoresis of the short (Figure 3, lane 1) and long (Figure 3, lane 2) T10 synthesis products. This result was confirmed by mass spectroscopy (18) (data not shown). Profiles of the products show an 80% increase in the amount of T10 product. Therefore, it is key to note that by alteration of only one parameter, the quantity of full-length DNA synthesized can be dramatically affected.
Figure 3. Oligonucleotide analysis short and long T10 coupling products were eluted from monohydroxysilane slides, labeled with ATP and analyzed. Radiolabeled short (lane 1) and long (lane 2) coupling products were electrophoresed on a 20% PAGE–7 M urea 1x TBE gel.
The synthesis products of chips with homopolymers (T12) were also compared to that of heteropolymers (M12) (Figure 4; for sequence, see Table 1). Gel analysis of the synthesized M12 (mixed base) oligonucleotides (Figure 4a, lane 2) revealed a dramatic decrease in the quantity of full-length product as compared to the amount of full-length product of T12 (Figure 4a, lane 1) and these profile differences were noted regardless of the type of chip substrate used. In an attempt to increase the amounts of full-length DNA synthesized, additional modifications were performed altering the parameters of coupling time (3-fold extension), exposure time (3-fold increase), and through the addition of protocol steps (i.e. Ar drying step) . Subsequent optimization of AGS parameters allowed for a greater proportion of full-length oligonucleotide production, as noted here during the production of (2) 41 base heteropolymers (M41a, M41b) (Figure 4b). The gel profile of the synthesis products from the ‘unoptimized’ chip (Figure 4b, lane 1) showed little to no evidence of a 41 base oligonucleotide. However, gel profile of the products from the ‘optimized’ chip (Figure 4b, lane 2) displayed a significant band of the correct size. Quantitation of the M41 gel profiles (Figure 4b, lanes 1 and 2), revealed a 100-fold increase in the amount of full-length product.
Figure 4. Homo- and Heteropolymer synthesis and optimization. All products were eluted from base-labile linker slides, electrophoresed on a 20% polyacrylamide–7 M urea 1x TBE gel . (a) Homopolymers (T12; lane 1) and heteropolymers (M12; lane 2). (b) Heteropolymers (M41a, M41b) pre-optimization (M41a, M41b) (lane 1) and post-optimization (M41a, M41b) (lane 2).
Biological application
To confirm identity and determine whether the eluted oligonucleotides were biologically functional, a limited gene assembly (Figure 5) was performed using non-amplified ssDNA oligonucleotides eluted directly from chip surface or amplified dsDNA (‘AACED’) oligonucleotides. For non-amplified oligonucleotides to be used for extension during gene assembly, it was necessary that their 3' ends retain a hydroxyl-group (–OH). To accomplish this, oligonucleotides were synthesized on chips containing a base-labile linker which, upon treatment with base (NH4OH), releases oligonucleotides with 3'-OH groups.
Figure 5. Elution and assembly of chip oligonucleotides. Oligonucleotides were obtained by (a) synthesis on base-labile or monohydroxysilane slides after cleavage and post-processing steps. (b) Assembly occurred with eluted oligonucleotides which had been designed to contain a region of overlap to allow for annealing and extension.
To ‘assemble’ DNA sequences, overlapping sense and reverse complementary oligonucleotides were designed (Table 1), and, through assembly and amplification protocols, a longer DNA sequence was formed. This method was chosen due to previous work from our laboratory which has shown that the products of various gene assembly protocols have inherent differences in error rates. In our hands the PCR assembly reactions were the most successful. The procedures relying on PCR yielded products with lower error rates (K. E. Richmond, manuscript in preparation) and therefore was the assembly method used in this work. The identity of the amplified products derived from the assembly of eluted oligonucleotides was confirmed through the addition of diagnostic restriction enzyme sites to the designed sequence. The foundation for oligonucleotide design was based on the sequence of Green Fluorescent Protein gene (GFPuv, Clontech).
‘Non-amplified’ oligonucleotides (chip eluates) were tested for their ability to assemble. Two (41 base) oligonucleotides (M41c, M41d) were synthesized and subsequently released from base-labile linkers for use in a minimal gene assembly process to form a 61 base fragment (Figure 6a, lane 1). The identity of the amplified assembled fragment was confirmed with a diagnostic digest (Figure 6a, lane 2) and analyzed on a denaturing polyacrylamide gel.
Figure 6. Application of chip oligonucleotides. (a) assembly of (2) eluted 41 base oligonucleotides; Radiolabeled assembly product digested with EcoRI (lane 1) and undigested (lane 2). Std: Oligo Ladder (Amersham) 8–32 bases; 60: a 60 base oligonucleotides standard. (b) Radiolabeled product of ‘Booster’ amplification (lane 1); product(lane 1) digested with PvuII (lane 2); product(lane 1) digested with MlyI (lane 3). Analysis of (a) and (b) 32P-labeled products on a 20% polyacrylamide–7 M urea 1x TBE gel. (c) Assembly of ‘AACED’ amplified product (lane 1); product (lane 1) digested with XhoI (lane 2). Std: 100 bp ladder (Promega).
We proposed that a possible drawback for using chip-eluted ssDNA as the direct input for some biological applications could be the inherently small concentrations available for use. To dramatically increase the overall amount of chip DNA, an amplification strategy called ‘amplification of assembled chip-eluted DNA’ was tested. AACED allows for the selection and amplification of full-length chip oligonucleotide populations, followed by the subsequent, enzyme-mediated removal of primer sequences flanking the unique internal DNA sequences (Table 1). The ability to obtain a diverse, large set of oligonucleotides in this manner may revolutionalize many biological applications since it provides access to the hundreds of thousands of user-defined sequences created on glass surfaces. In our experiments, the restriction enzyme MlyI was designed into upstream and downstream primer sequences to allow for complete blunt removal of primer sequences post-amplification. Therefore, after producing high copy numbers of the full oligonucleotides, the flanking primer sequences are removed, yielding the variable internal oligonucleotide sequences ready for use in any number of applications.
As a proof of concept, a single 60 base oligonucleotide was synthesized, eluted, amplified (Figure 6b, lane1) and digested with MlyI (Figure 6b, lane 3) to allow for the release of the upstream and downstream flanking 15 base primer sequences, leaving a single 30 base oligonucleotide for subsequent applications. The digested sample was then labeled using ATP and T4 polynucleotide kinase and analyzed on a denaturing polyacrylamide gel. The amplified product from the chip eluate (Figure 6b, lane 1) gave a strong single band of the correct size (60 bp) which digested as expected with the diagnostic restriction enzyme (Figure 6b, lane 2). To determine whether the MlyI segments were efficiently released, the digested sample was also radiolabeled and analyzed (Figure 6b, lane 3) and the only band evident was a doublet of the correct size (15 bp). This doublet is postulated to occur due to the slight differences in molecular weight between the released, mixed-base 15 base fragments. Due to the presence of 5' phosphates, the internal 30 base fragments were not labeled and therefore not visible in the lane containing the Mly1 digestion (Figure 6b, lane 3). Of note is the lack of the initial 60 base band (Figure 6b, lane 3), indicating that all the input DNA was thoroughly digested with MlyI.
With the ‘AACED’ concept verified, larger assemblies of eight overlapping sense and reverse complement oligonucleotides were performed (Figure 6c). The synthesized oligonucleotides (Table 1) were 70 bases in length, with the initial and final 15 bases designed for primer sequences, leaving an internal 40 base sequence for subsequent use in assembly. Analysis of the amplified assembly product revealed a band of the expected assembled product size (180 bp) (Figure 6c, lane 1) and its identity was confirmed by a diagnostic digest (Figure 6c, lane 2).
DISCUSSION
Prior to performing this analysis of chip-eluted oligonucleotides, modifications of the maskless array synthesizer were performed to allow for high-quality, high-density synthesis of long oligonucleotides onto glass slides. The enhancements utilized existing technology such as an image-locking system to allow for the improved, accurate synthesis of long oligonucleotides up to 90 bases in length, as shown in imaging and fluorescence studies . This technique allows for the synthesis of not only longer oligonucleotides, but also more accurate oligonucleotides (by reducing inaccurate deprotection which causes subsequent base addition and/or deletion), and the ability to create more sequences per chip (reducing the need for ‘border’ areas to limit crossover deprotection of adjoining regions from drift).
One significant finding was the dramatic effect noted by the alteration of a single synthesis parameter on the quantity of full-length oligonucleotides produced. It is evident that the ‘standard’ method for DNA synthesis, although adequate for microarray production and use, may not be the best manner in which to synthesize large quantities of full-length oligonucleotides. Analysis of the synthesis products of the homopolymer T10 (Figure 3, lane 2) revealed that only a minority of oligonucleotides were full length (<35% of population). This was interesting since the coupling efficiency for NPPOC-T had been reported previously (98%) and for a T10, therefore, 76% of products were expected to be full length. Such differences between the theoretical and experimental full-length oligonucleotides production could be accounted for by a myriad of reasons such as inefficiencies in deprotection (4), coupling, or cleavage from the chip surface. Prior to this work, no analysis of chip eluates had been done on chips generated with NPPOC chemistry. These results led us to optimize the DNA synthesis parameters (coupling, exposure time, protocol steps) used in this study.
The difference in profiles between mixed base and poly(T) oligonucleotides (Figure 4a) was significant and intriguing. Although the efficiencies of base addition have been reported previously (11) and shown to vary for the different NPPOC phosphoramidites (A-96%, G-97%, T-98% and C-99%), it is unlikely that these differences alone account for the decrease in M12 full-length products. Further optimization studies were performed to increase the amount of full-length product and it was noted that a 100-fold increase occurred not by changing the basic chemistry or reagents used, but through changes in the synthesis protocol itself. The combination of increased coupling time (from 20 to 60 s), increased exposure time(from 50 to 150 s), the addition of Ar drying steps, and an image-locking system was used to produce more full-length product even for longer oligonucleotides such as 40mers (Figure 4b).
Interestingly, chips that were not optimized for DNA synthesis yielded strong hybridization profiles (data not shown). Despite the lack of full-length oligonucleotides seen in gel profiles, hybridization appears sensitive enough to detect these species, present in only small percentages, and, therefore, hybridization results do not accurately determine overall oligonucleotide quality; they are merely indicative of trends in purity. In high-density arrays there are also additional factors which can affect the interpretation of hybridization results and make these results unreliable for any predicative use for chip oligonucleotide quality.
Initial experiments with oligonucleotides (41mers) from ‘optimized’ syntheses on base-labile slides were successful in assembling to form a larger DNA fragment (61mers). This is the first reported use of the base-labile linkage for release of DNA from chips and the use of eluted chip DNA in a biological application. Assemblies using amplified and digested oligonucleotides (eight 70mers digested to yield eight 40mers) were also successful and this method could be applied to create large populations of oligonucleotides for other biological purposes. The ‘AACED’ amplifying method has the advantage of not only increasing the number of oligonucleotides for use, but it also allows for the enrichment of the full-length product over truncate species. Additionally this method could allow for amplification of sub-populations of DNA from the chip surface, making each chip useful for a variety of oligonucleotides syntheses. Indeed, with the (overly) optimistic assumption of obtaining 10 pmol/cm2 of oligonucleotides from a chip, this quantity would correspond to 12 attomol/pixel and thus without amplification, most biological applications would not be feasible. If our AACED was extended to its full potential, a single chip with 786 432 unique 40mer oligonucleotides could potentially be used to assemble >15 Mb of DNA.
Miniaturization of hardware provides a means to reduce the quantities of reagents and therefore the costs of oligonucleotide synthesis. Technological applications using oligonucleotides, however, are becoming very diverse and hence the variety of sequences needed is escalating. In keeping with this trend, we have utilized a Biological Exposure Synthesis System (BESS) to produce chips with small amounts of synthesized oligonucleotides for subsequent elution and utilization in biological assays. This system can allow for hundreds of thousands of discrete oligonucleotides species to be made inexpensively and quickly for utilization in a myriad of biological assays, including that of gene assembly.
ACKNOWLEDGEMENTS
We would like to acknowledge that this work was supported by DARPA Grant No. DAAD 19-02-2-0026.
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