High-Throughput Site-Directed Mutagenesis using Oligonucleotides Synthesized on DNA Chips

Abstract

Site-directed mutagenesis has greatly helped researchers both to understand the precise role of specific residues in coding sequences and to generate variants of proteins that have acquired new characteristics. Today's demands for more complete functional cartographies of proteins and advances in selection and screening technologies require that site-directed mutagenesis be adapted for high-throughput applications. We describe here the first generation of a library of single and multiple site-directed mutants using a mixture of oligonucleotides synthesized on DNA chips. We have used the human interleukin 15 (IL15) gene as a model, of which 37 codons were simultaneously targeted for substitution by any of eight possible codons. Ninety-six clones were sequenced, exhibiting a broad spectrum of targeted substitutions over the whole gene length with no unwanted mutations. Libraries produced using such pools of oligonucleotides open new perspectives to direct the evolution of proteins in vitro, by enabling the simple, rapid, and cost-effective generation of large tailor-made genetic diversities from any gene.

Photoelectrochemical synthesis of DNA microarrays

“…The large synthesis capacity of the porous glass film may greatly benefit applications that use custom microarrays to drive down the cost per base for genomic research, such as multiplexed gene synthesis (16,26,36), library construction (37,38), and genomic selection for targeted sequencing (39)(40)(41). It effectively further lowers the cost per base by reducing the spot redundancy required to obtain biologically relevant quantities of each sequence, and also limits the need for amplification protocols.…”

Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process

“…The main methods used to achieve spatial control include (i) control of the coupling step by inkjet printing (Agilent, Protogene) (12,13) or physical masks (Southern) (14), (ii) control of the 5′-hydroxyl deblock step by classical (Affymetrix) (15) and maskless (Nimblegen) (16) photolithographic deprotection of photolabile monomers or (iii) digital activation of photogenerated acids to carry out standard detritylation (Xeotron/Atactic) (17). More recently, oligonucleotides made on commercial microarrays have been cleaved from their solid surface and pooled to enable new applications such as shRNA libraries (18), gene synthesis (19,20) and site-directed mutagenesis (21). While there are other avenues to create pools of oligonucleotides of length <100mer (22,23), microarray-based synthesis of oligonucleotides is a method particularly well adapted to any application that requires minute amounts of thousands of different DNA sequences in a cost effective manner.…”

Improvement of an Antibody Neutralizing the Anthrax Toxin by Simultaneous Mutagenesis of Its Six Hypervariable Loops

“…3), described in Ref. 26 The resulting mixture was subjected to 12 temperature cycles (at 94°C for 1 min, 50°C for 2 min, and 68°C for 20 min). Four microliters of 10× buffer 4 (New England Biolabs), 0.5 μl of DpnI (20 U/μl; New England Biolabs), and 15.5 μl of deionized water were added to the 25-μl final volume of the previously amplified products and incubated for 30 min at 37°C.…”