By-Product Formation in Repetitive PCR Amplification of DNA Libraries during SELEX

The selection of nucleic acid aptamers is an increasingly important approach to generate specific ligands binding to virtually any molecule of choice. However, selection-inherent amplification procedures are prone to artificial by-product formation that prohibits the enrichment of target-recognizing aptamers. Little is known about the formation of such by-products when employing nucleic acid libraries as templates. We report on the formation of two different forms of by-products, named ladder- and non-ladder-type observed during repetitive amplification in the course of in vitro selection experiments. Based on sequence information and the amplification behaviour of defined enriched nucleic acid molecules we suppose a molecular mechanism through which these amplification by-products are built. Better understanding of these mechanisms might help to find solutions minimizing by-product formation and improving the success rate of aptamer selection.     

金黄色葡萄球菌外毒素B特异性适体的筛选及其应用

目的:利用指数富集配基的系统进化(SELEX)技术,筛选能与金黄色葡萄球菌外毒素B(SEB)特异、高亲和力结合的单链DNA(ssDNA)适体,并将该适体应用于患者血清标本的检测。方法:从体外合成的96核苷酸随机ss-DNA文库中,以羧基磁珠作为筛选介质,经逐步PCR扩增、筛选,获得针对SEB的高亲和力、高特异性适体;利用荧光素标记适体测定筛选过程中各轮结合力;利用酶连接适体方法检测适体特异性和结合力。结果:经过13轮筛选,ssDNA文库与SEB的结合百分率从1.1%提高到39.8%,增加了36倍;获得的ssDNA适体(A11)针对SEB的特异性强,与金黄色葡萄球菌表面蛋白A(SPA)结合低,并能初步识别患者血清。结论:利用SELEX技术筛选获得了特异结合SEB的高亲和力的ssDNA适体,为金黄色葡萄球菌的临床诊断与治疗奠定了基础。     

Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment

Oligonucleotide aptamers represent a novel platform for creating ligands with desired specificity, and they offer many potentially significant advantages over monoclonal antibodies in terms of feasibility, cost, and clinical applicability. However, the isolation of high-affinity aptamer ligands from random oligonucleotide pools has been challenging. Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool. We show that emulsion PCR preserves library diversity, preventing the loss of rare high-affinity aptamers that are difficult to amplify. We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity. Using a combination of bioinformatics and functional analyses, we show that the rate of amplification is more predictive than prevalence with respect to binding affinity and that the mutational landscape within a cluster of related aptamers can guide the identification of high-affinity aptamer ligands. Finally, we demonstrate the power of this selection process for identifying cross-species aptamers that can bind human receptors and cross-react with their murine orthologs.     

Use of magnetic beads in selection and detection of biotoxin aptamers by electrochemiluminescence and enzymatic methods.

Systematic evolution of ligands by exponential enrichment (SELEX) was used to develop DNA ligands (aptamers) to cholera whole toxin and staphylococcal enterotoxin B (SEB). Affinity selection of aptamers was accomplished by conjugating the biotoxins to tosyl-activated magnetic beads. The use of magnetic beads reduces the volumes needed to perform aptamer selection, thus obviating alcohol precipitation and allowing direct PCR amplification from the bead surface. Following five rounds of SELEX, 5'-biotinylated aptamers were bound to streptavidin-coated magnetic beads and used for the detection of ruthenium trisbypyridine [Ru(bpy)3(2+)]-labeled cholera toxin and SEB by an electrochemiluminescence methodology. A comparison of control (double-stranded) aptamer binding was made with aptamers that were heat denatured at 96 degrees C (single-stranded) and allowed to cool (conform) in the presence of biotoxin-conjugated magnetic beads. Results suggest that control aptamers performed equally well when compared to heat-denatured DNA aptamers in the cholera toxin electrochemiluminescence assay and a colorimetric microplate assay employing peroxidase-labeled cholera toxin and 5'-amino terminated aptamers conjugated to N-oxysuccinimide-activated microtiter wells. Interestingly, however, in the SEB electrochemiluminescence assay, double-stranded aptamers exceeded the performance of single-stranded aptamers. The detection limits of all aptamer assays were in the low nanogram to low picogram ranges.     

Advances in SELEX and application of aptamers in the central nervous system

SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a screening technique that involves the progressive selection of highly specific ligands by repeated rounds of partition and amplification from a large combinatorial nucleic acid library. The products of the selection are called aptamers, which are short single stranded DNA or RNA molecules, binding with high affinity, attributed to their specific three-dimensional shapes, to a large variety of targets, ranging from small molecules to complex mixtures. Various improvement of the original SELEX method described in 1990 have been obtained recently, such as capillary electrophoresis SELEX, Toggle-SELEX, Tailored-SELEX, Photo-SELEX, and others. These new variants greatly shorten time of selection and improve aptamer affinity and specificity. Such aptamers have great potential as detecting and/or diagnostic reagents. Furthermore, some aptamers specifically inhibit biological functions of targeted proteins, and are considered as potent therapeutic lead structures evaluated in preclinical disease models. Recently, one aptamer has been approved by Food and Drug Administration of US for treating age-related macular degeneration. This review presents recent advances in the field of SELEX with special emphasis on applications of aptamers as analytical, diagnostic and therapeutic tools in the central nervous system.     

分子模拟指导核酸适配体文库设计

核酸适配体是利用配体指数富集的系统进化技术（SELEX）从随机文库中筛选获得一段有功能的单链寡核苷酸。但因筛选过程中的文库选择、洗涤次数、分离效率、缓冲液离子含量和pH值等多种因素的影响,迄今所报道的亲和力与特异性都很高的核酸适配体为数不多。初始文库是核酸适配体筛选的源头,作为SELEX技术的根本,其设计是否合理直接影响到筛选的成败和效率。分子模拟能以核酸适配体文库为主体,计算机为主要工具,发展多种结构模拟与分析工具,辅助核酸适配体文库的合理设计。本文综述了现阶段利用分子模拟进行核酸适配体初始文库设计的相关方法,希望能为从源头上提高核酸适配体筛选的成功率提供线索。     

两种富集方法相结合对蓖麻毒素进行SELEX筛选研究

为了获得能特异识别具有细胞毒性的蓖麻毒素蛋白寡核苷酸适配子,体外构建了含40个随机序列全长87nt的随机ssDNA文库,采用指数富集配基的系统进化(SELEX)技术方法,结合微孔板和亲和树脂两种分离、富集方法,经过数轮筛选,文库与蓖麻毒素的结合率达到了38．5％。结果表明,以亲和树脂为分离介质进行筛选,富集效果非常明显。     

Construction of low melting point agarose emulsion PCR amplification complex gene sequence

Emulsion polymerase chain reaction, an effective amplification, can make millions of templates could be individually amplified within a single tube. Here we constructed and improved a low melting point agarose-emulsion method to promote the specific sequences amplification effectively. Artificial Lactobacillus Plasmid as template was amplified and clear fluorescence images of the agarose beads were obtained. The Real-time PCR data showed that agarose-emulsion PCR clearly indicated that DNA can be amplified in agarose droplets. Overall, our study effectively overcame the difficulty of formation of uniform emulsion droplets, negative effect on recombination of homologous regions of DNA and generation of void emulsion droplets. This method increases the accuracy with amplification, reduces the influence of uncertainties and provides the reliable data for further experiment.     

Minimizing DNA contamination by using UNG-coupled quantitative real-time PCR on degraded DNA samples: application to ancient DNA studies

PCR analyses of ancient and degraded DNA suffer from their extreme sensitivity to contamination by modern DNA originating, in particular, from carryover contamination with previously amplified or cloned material. Any strategy for limiting carryover contamination would also have to be compatible with the particular requirements of ancient DNA analyses. These include the need (i) to amplify short PCR products due to template fragmentation; (ii) to clone PCR products in order to track possible base misincorporation resulting from damaged templates; and (iii) to avoid incomplete decontamination causing artifactual sequence transformation. Here we show that the enzymatic decontamination procedures based upon dUTP- and uracil-N-glycosylase (UNG) can be adapted to meet the specific requirements of ancient DNA research. Thus, efficiency can be improved to vastly reduce the amplification of fragments < or = 100 bp. Secondly, the use of an Escherichia coli strain deficient in both UNG and dUTPase allows for the cloning of uracil-containing PCR products and offers protection from plasmid DNA contamination, and, lastly, PCR products amplified from UNG-degraded material are free of misleading sequence modifications.     

Non-SELEX: selection of aptamers without intermediate amplification of candidate oligonucleotides

Aptamers are typically selected from libraries of random DNA (or RNA) sequences through systematic evolution of ligands by exponential enrichment (SELEX), which involves several rounds of alternating steps of partitioning of candidate oligonucleotides and their PCR amplification. Here we describe a protocol for non-SELEX selection of aptamers--a process that involves repetitive steps of partitioning with no amplification between them. Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), which is a highly efficient affinity method, is used for partitioning. NECEEM also facilitates monitoring of bulk affinity of enriched libraries at every step of partitioning and screening of individual clones for their affinity to the target. NECEEM allows all clones to be screened prior to sequencing, so that only clones with suitable binding parameters are sequenced. The entire protocol can be completed in 1 wk, whereas conventional SELEX protocols take several weeks even in a specialized industrial facility.     

Selection of a DNA aptamer that binds 8-OHdG using GMP-agarose

DNA aptamers, which bind specific molecule, such as 8-OHdG, with high affinity were investigated using an in vitro selection strategy called systematic evolution of ligands by exponential enrichment (SELEX). However, 8-OHdG was difficult to immobilize on a carrier for SELEX. Therefore, a DNA aptamer binding to 8-OHdG was selected using GMP-agarose as an analogue from a library of about 4⁶⁰ random ssDNA sources. As a result, three aptamer candidates were selected. Among the selected DNA aptamers, the No. 22 DNA aptamer exhibited a high affinity for 8-OHdG. The dissociation constant, K_D, of No. 22 DNA aptamer was on the order of 0.1 μmol/L. This result suggests that using an analogue will be a useful new SELEX method for obtaining various aptamers that are difficult to immobilize on a matrix.     

Uniform amplification of multiple DNAs by emulsion PCR

When several DNAs are amplified by PCR in one PCR tube, biased amplification is known to occur because amplification efficiency differs from one DNA to another. Therefore, we conducted PCR in the water in oil-emulsion (W/O emulsion) to examine whether the procedure allows the uniform amplification of several DNAs. In the amplification of a model library consisting of two clones, the emulsification of the PCR mixture successfully reduced the difference in its amplification efficiency to approximately one-seventh the value obtained without emulsification. Furthermore, we conducted repeated PCR to amplify a model library consisting of ten short hairpin RNA (shRNA) expression vectors as a model experiment for gene discovery using an shRNA expression library. Consequently, the emulsification of the PCR mixture successfully reduced PCR bias. Our results indicate that emulsion PCR is capable of uniformly amplifying libraries of shRNA, ribozyme, cDNA, and others, and is useful also for gene discovery using these libraries.     

Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments

We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.     

Indirect purification method provides high yield and quality ssDNA sublibrary for potential aptamer selection

The quality and yield of single-stranded DNA (ssDNA) play key roles in ssDNA aptamer selection. However, current methods for generating and purifying ssDNA provides either low yield due to ssDNA loss during the gel purification process or low specificity due to tertiary structural damage of ssDNA by alkaline or exonuclease treatment in removing dsDNA and by-products. This study developed an indirect purification method that provides a high yield and quality ssDNA sublibrary. Symmetric PCR was applied to generate a sufficient template, while asymmetric PCR using an excessive nonbiotinylated forward primer and an insufficient biotinylated reverse primer combined with a biotin–strepavidin system was applied to eliminate dsDNA, hence, leading to ssDNA purification. However, no alkaline or exonuclease were involved in treating dsDNA, so as to warrant the tertiary structure of ssDNA for potential aptamer SELEX selection. Agarose gel imaging indicated that no dsDNA or by-product contamination was detected in the ssDNA sublibrary generated by the indirect purification method. Purified ssDNA concentration reached 1020 ± 210 nM, which was much greater than previous methods. In conclusion, this novel method provided a simple and fast tool for generating and purifying a high yield and quality ssDNA sublibrary.     

A rapid reliable method to use specific probes labelling polymerase chain reaction (PCR) products.

Polymerase chain reaction (PCR) has allowed highly sensitive detection and amplification of individual DNA sequences. To generate specific probes for genes or cDNAs that have not yet been cloned, it is often necessary to label PCR products which are then used in Southern or Northern hybridizations or for screening cDNA and genomic DNA libraries. In this paper a rapid and versatile method of using PCR products, as specific probes, is described, after digestion with EcoRI in buffer H, in the presence of PCR reaction buffer, and purification of the PCR products for avoid the interference by competition of unlabelled dCTP in the directionally random labelling.     

The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: application to in vitro combinatorial selection of aptamers

A PCR method for uniform amplification of a random sequence DNA library is described. A combination of 1 M betaine and 5% DMSO improves the PCR amplification by increasing the ratio of full-length products to shortened products, which are a consequence of nonuniform amplification due to stable secondary structures in the templates. This method is expected to be beneficial for obtaining high-affinity aptamers with stable secondary structures.     

Expanding the application potential of DNA aptamers by their functionalization

Side-by-side development of two competing technologies for obtaining affinity antibody-based and aptamer-based molecules opens new horizons for the creation of diagnostic and therapeutic agents of extremely high efficiency. Benefits of aptamers, such as relatively small size and selection simplicity, have been jeopardized for a long time by their intrinsic downsides, i.e., obscure process of obtaining aptamers against certain targets because of a low diversity of functional groups (purine and pyrimidine bases) in DNA and RNA aptamers. Another side effect of the aptamer technique inherent to the traditional SELEX method is unspecific enrichment with aptamers with high affinity to off-target reaction components. Today, due to current progress in the development of new technology methods and chemical coupling reactions, the modern aptamer technology helps to avoid its disadvantages and become capable of being the source of new diagnostic and therapeutic tools, which are properly unique in their efficiency. The review focuses on modern methods of increasing efficiency of the aptamer selection and on synthetic nucleotide modifications, which make it possible to prepare high-affinity aptamers against traditionally ‘hard’ targets.     

Comparison between Taq DNA polymerase and its Stoffel fragment for quantitative real-time PCR with hybridization probes

In quantitative real-time PCR assays, fluorophor-labeled oligonucleotide probes are employed to generate sequence-specific signals for the quantitative evaluation. Whereas TaqMan probes have to be hydrolyzed during PCR by the endonucleolytic activity of Taq DNA polymerase to generate a signal, the hybridization probes in LightCycler assays must not be hydrolyzed. In this study, we demonstrate for four different targets that the probes are degraded during PCR by Taq DNA polymerase. Signal yield, quality of amplification curves, and accuracy of quantitative measurements can be improved using the Stoffel fragment lacking an endonucleolytic activity and TaqStart antibody suppressing the formation of nonspecific products, without laborious efforts to optimize the amplification protocol.