Directed evolution of nucleotide-based libraries using lambda exonuclease

Directed evolution of nucleotide libraries using recombination or mutagenesis is an important technique for customizing catalytic or biophysical traits of proteins. Conventional directed evolution methods, however, suffer from cumbersome digestion and ligation steps. Here, we describe a simple method to increase nucleotide diversity using single-stranded DNA (ssDNA) as a starting template. An initial PCR amplification using phosphorylated primers with overlapping regions followed by treatment with lambda exonuclease generates ssDNA templates that can then be annealed via the overlap regions. Double-stranded DNA (dsDNA) is then generated through extension with Klenow fragment. To demonstrate the applicability of this methodology for directed evolution of nucleotide libraries, we generated both gene shuffled and regional mutagenesis synthetic antibody libraries with titers of 2x108 and 6x107, respectively. We conclude that our method is an efficient and convenient approach to generate diversity in nucleic acid based libraries, especially recombinant antibody libraries

Probing the SELEX Process with Next-Generation Sequencing

Background SELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large part of the population dynamics during the selection process. Methodology We have performed a semi-automated SELEX procedure on the model target streptavidin starting with a synthetic DNA oligonucleotide library and compared results obtained by the conventional analysis via cloning and Sanger sequencing with next-generation sequencing. In order to follow the population dynamics during the selection, pools from all selection rounds were barcoded and sequenced in parallel. Conclusions High affinity aptamers can be readily identified simply by copy number enrichment in the first selection rounds. Based on our results, we suggest a new selection scheme that avoids a high number of iterative selection rounds while reducing time, PCR bias, and artifacts

Automation in the high-throughput selection of random combinatorial libraries-different approaches for select applications

Automation in combination with high throughput screening methods has revolutionised molecular biology in the last two decades. Today, many combinatorial libraries as well as several systems for automation are available. Depending on scope, budget and time, a different combination of library and experimental handling might be most effective. In this review we will discuss several concepts of combinatorial libraries and provide information as what to expect from these depending on the given context

Mol Cell Proteom

Due to the success of DNA microarrays and the growing numbers of available protein expression clones, protein microarrays have become more and more popular for the high throughput screening of protein interactions. However, the widespread applicability of protein microarrays is currently hampered by the large effort associated with their production. Apart from the requirement for a protein expression library, expression and purification of the proteins themselves and the lacking stability of many proteins remain the bottleneck. Here we present an approach that allows the generation of high density protein microarrays from unbound DNA template molecules on the chip. It is based on the multiple spotting technique and comprises the deposition of a DNA template in a first spotting step and the transfer of a cell-free transcription and translation mixture on top of the same spot in a second spotting step. Using wild-type green fluorescent protein as a model protein, we demonstrated the time and template dependence of this coupled transcription and translation and showed that enough protein was produced to yield signals that were comparable to 300 µg/ml spotted protein. Plasmids as well as unpurified PCR products can be used as templates, and as little as 35 fg of PCR product (~22,500 molecules) were sufficient for the detectable expression of full-length wild-type green fluorescent protein in subnanoliter volumes. We showed that both aminopropyltrimethoxysilane and nickel chelate surfaces can be used for capture of the newly synthesized proteins. Surprisingly we observed that nickel chelate-coated slides were binding the newly synthesized proteins in an unspecific manner. Finally we adapted the system to the high throughput expression of libraries by designing a single primer pair for the introduction of the required T7 promoter and demonstrated the in situ expression using 384 randomly chosen clones

Trends Mol Med

High-throughput protein arrays allow the miniaturized and parallel analysis of large numbers of diagnostic markers in complex samples. Using automated colony picking and gridding, cDNA or antibody libraries can be expressed and screened as clone arrays. Protein microarrays are constructed from recombinantly expressed, purified, and yet functional proteins, entailing a range of optimized expression systems. Antibody microarrays are becoming a robust format for expression profiling of whole genomes. Alternative systems, such as aptamer, PROfusion™, nano- and microfluidic arrays are all at proof-of-concept stage. Differential protein profiles have been used as molecular diagnostics for cancer and autoimmune diseases and might ultimately be applied to screening of high-risk and general populations. Abstract 2 : High-throughput protein arrays are still largely experimental but have taken the first steps towards becoming diagnostic tools, which will eventually arrive at the doctor's practice and as over-the-counter devices

Next generation of protein microarray support materials: evaluation for protein and antibody microarray applications

The performance of protein and antibody microarrays is dependent on various factors, one of which is the use of an appropriate microarray surface for the immobilisation of either protein or antibody samples. We have investigated the properties of seven new surfaces in the context of both protein and antibody microarray technology. We have demonstrated the functionality of all new slide coatings and investigated the mean signal to spotted concentration ratio, determined detection limits and calculated coefficients of variation. Moreover, new concepts for slide coatings such as dendrimer and poly(ethylene glycol)-epoxy slides were evaluated and improved qualities of novel slide surfaces were observed. Optimal slide coatings for antibody and protein chips were proposed and the requirements for both technologies were discussed

J. Chromatogr. A

The performance of protein and antibody microarrays is dependent on various factors, one of which is the use of an appropriate microarray surface for the immobilisation of either protein or antibody samples. We have investigated the properties of seven new surfaces in the context of both protein and antibody microarray technology. We have demonstrated the functionality of all new slide coatings and investigated the mean signal to spotted concentration ratio, determined detection limits and calculated coefficients of variation. Moreover, new concepts for slide coatings such as dendrimer and poly(ethylene glycol)-epoxy slides were evaluated and improved qualities of novel slide surfaces were observed. Optimal slide coatings for antibody and protein chips were proposed and the requirements for both technologies were discussed