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. 2004 Sep 24;32(17):5011-8.
doi: 10.1093/nar/gkh793. Print 2004.

Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis

Kathryn E Richmond  1 , Mo-Huang Li, Matthew J Rodesch, Madhusudan Patel, Aaron M Lowe, Changhan Kim, Larry L Chu, Narasimhar Venkataramaian, Shane F Flickinger, James Kaysen, Peter J Belshaw, Michael R Sussman, Franco Cerrina
Affiliations

Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis

Kathryn E Richmond et al. Nucleic Acids Res. .

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.

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Figures

Figure 1
Figure 1
Biological Exposure and Synthesis System (BESS).
Figure 2
Figure 2
Chemical synthesis of a novel base labile linker.
Figure 3
Figure 3
Oligonucleotide analysis short and long T10 coupling products were eluted from monohydroxysilane slides, labeled with [γ-32P]ATP and analyzed. Radiolabeled short (lane 1) and long (lane 2) coupling products [L: oligonucleotides ladder 8–32 bp (Amersham)] were electrophoresed on a 20% PAGE–7 M urea 1× TBE gel.
Figure 4
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 1× TBE gel [Std: Oligonucleotide ladder, 8–32 bases (Amersham)]. (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).
Figure 5
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.
Figure 6
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 1× TBE gel. (c) Assembly of ‘AACED’ amplified product (lane 1); product (lane 1) digested with XhoI (lane 2). Std: 100 bp ladder (Promega).
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