Cost-effective interrogation of single nucleotide polymorphisms using the mismatch amplification mutation assay and capillary electrophoresis

The ability to characterize SNPs is an important aspect of many clinical diagnostic, genetic and evolutionary studies. Here, we designed a multiplexed SNP genotyping method to survey a large number of phylogenetically informative SNPs within the genome of the bacterium Bacillus anthracis. This novel method, CE universal tail mismatch amplification mutation assay (CUMA), allows for PCR multiplexing and automatic scoring of SNP genotypes, thus providing a rapid, economical and higher throughput alternative to more expensive SNP genotyping techniques. CUMA delivered accurate B. anthracis SNP genotyping results and, when multiplexed, saved reagent costs by more than 80% compared with TaqMan real-time PCR. When real-time PCR technology and instrumentation is unavailable or the reagents are cost-prohibitive, CUMA is a powerful alternative for SNP genotyping.     

Single-Base Extension and ELISA-Based Approach for Single-Nucleotide Polymorphisms Genotyping

Single-nucleotide polymorphisms (SNPs) emerge as a fundamental tool in personalized medicine due to their association with drug responses or disease predisposition. Single-base extension (SBE) is a common method for characterizing known SNPs, but involves complicated procedures or requires costly analytical instruments. Here, we describe a novel SNP genotyping based on SBE and enzyme-linked immunosorbent assay (ELISA). During the SBE, the 5′ end fluorescein isothiocyanate-labeled allele-specific primer will extend with biotinylated dideoxynucleotides which are complementary to the SNP sites. The extension product will then be captured by streptavidin-coated nanoparticle and develop blue color in the ELISA assay. We validated this method by detecting SNPs for TP53 gene codon 273 from 68 individuals and the data were 100% in concordant with DNA sequencing. Thus, SBE and ELISA-based SNPs assay is a simple and accurate method for SNP genotyping.     

Label-free genotyping of single-nucleotide polymorphisms for DNA and RNA targets using a cationic polythiophene derivate

By using the specific primer extension reaction, a new assay for genotyping of single-nucleotide polymorphisms (SNPs) has been demonstrated. The assay relies on the conformational and colorimetric change of water-soluble polythiophene derivative, poly[3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydrochloride] (PMNT), upon forming interpolyelectrolyte complex with extended double strand DNA and non-extended single strand DNA. All three kinds of SNP genotypes can be colorimetrically identified with one primer extension reaction in homogeneous solution. Moreover, combining with the specific digestion of RNA strands in the RNA/DNA hybrids, the proposed assay can also be applied to SNP genotyping for RNA templates. The SNP genotyping assay does not require chemical modification of oligonucleotide probes and nucleic acid targets and any separation step. It would be useful for routinely SNP detection in ordinary laboratories.     

Improved adapter-ligation-mediated allele-specific amplification for multiplex genotyping by using software

Adapter-ligation-mediated allele-specific amplification (ALM-ASA) is a potential method for multiplex SNPs typing at an ultra low cost. Here, we describe a kind of software, which designs allele-specific primers for ALM-ASA assay on multiplex SNPs. DNA sequences containing SNPs of interest are submitted into the software which contains various endonucleases for options. Based on the SNP sequence information and the selected endonucleases, the software is capable of automatically generating sets of information needed to perform genotyping experiments. Each set contains a suitable endonuclease, qualified allele-specific primers with orientations and melting temperatures, sizes of allele-specific amplicons, and gel electropherograms simulated according to the sizes of the allele-specific amplicons and the mobility of DNA fragments in 2% agarose gel. Seven SNPs in the arylamines N-acetyltransferase 2 (NAT2) gene, five SNPs in the BRCA1 gene, five SNPs in the COMT gene, six SNPs in the CYP2E1 gene, five SNPs in the MPO gene, and six SNPs in the NRG1 gene were selected for evaluating the software. Without extra optimization, seven SNPs in the NAT2 gene were successfully genotyped for genomic DNA samples from 127 individuals by using the first set of allele-specific primers yielded by the software. Although several steps are used in the ALM-ASA assay, the whole genotyping process can be completed within 3 h by optimizing each step. Profiting from the software, the ALM-ASA assay is easy-to-perform, labor-saving, and accurate.     

Detection of single nucleotide polymorphisms using electrospray ionization mass spectrometry: validation of a one-well assay and quantitative pooling studies

Single nucleotide polymorphisms (SNPs) are currently being mapped and databased at a remarkable pace, providing a viable means for understanding disease susceptibility, differential drug response and human evolution. Consequently, there is an increasing demand for SNP genotyping technologies that are simple, rapid, cost effective and readily amenable to automation for high-throughput analyses. In this study, we improved the Survivor Assay, a SNP detection method based on electrospray ionization mass spectrometry (ESI-MS), with several developments. One improvement is the development of a one-well assay, requiring no off-line purification of the polymerase chain reaction product, achieved by simple addition of reagent solution into a single well. Another is the on-line separation of magnesium and dideoxynucleotides using an in-house made monolithic metal chelating column, eliminating any off-line sample preparation prior to mass spectrometric analysis. Here the Survivor Assay is extended from a proof-of-principle concept to a validated method by genotyping six SNPs from five different regions of human genomic DNA in 55 individual samples with 100% accuracy. This improved Survivor Assay eliminates the tedious and time-consuming steps of sample preparation, minimizes sample handing and offers a high-throughput analysis of SNPs by ESI-MS. The current combined preparation and analysis time is 2 min per sample. The simplicity of this method has potential for full automation and parallel chromatography and, thus, reduced analysis time. In addition, we have adapted the Survivor Assay for quantitative SNP analysis in pooled DNA samples. The capabilities and sensitivity of this approach were evaluated. We demonstrate that an allele occurring at a frequency of 2% can consistently be quantitated.     

A standard protocol for single nucleotide primer extension in the human genome using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Analysis of single nucleotide polymorphisms (SNPs) has become an increasingly important area of research, with numerous applications in medical genetics, population genetics, forensic science, and agricultural biotechnology. Large-scale SNP analyses require the development of methodologies that are economical, flexible, accurate and capable of automation. Primer extension in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is currently emerging as a potential method for high-throughput SNP genotyping. We have evaluated a number of published primer extension methods and refined a simple and robust protocol to analyze human autosomal disease-causing mutations and population genetic markers on the Y-chromosome. Twelve different variant sites were examined, and homozygotes, heterozygotes and hemizygotes were accurately typed. A 100% concordance was observed between SNP genotypes obtained using the MALDI-TOFMS technique and alternative genotyping methods, such as restriction fragment length polymorphism (RFLP) assays and denaturing high-performance liquid chromatography (DHPLC). Since multiple polymorphisms can be detected in single reactions, the method provides a cost-effective approach for SNP analysis. The protocol is also extremely flexible (able to accommodate new markers) and can be adapted to a number of platforms without the use of commercial kits.     

A multiplex SNP genotyping by allele‐specificspecific PCR based on stem‐loop and universal fluorescent primers of Chr1daxin mice

Single nucleotide polymorphisms (SNPs) are one of the most common markers in mammals. Rapid, accurate, and multiplex typing of SNPs is critical for subsequent biological and genetic research. In this study, we have developed a novel method for multiplex genotyping SNPs in mice. The method involves allele‐specific PCR amplification of genomic DNA with two stem‐loop primers accompanied by two different universal fluorescent primers. Blue and green fluorescent signals were conveniently detected on a DNA sequencer. We verified four SNPs of 65 mice based on the novel method, and it is well suited for multiplex genotyping as it requires only one reaction per sample in a single tube with multiplex PCR. The use of universal fluorescent primers greatly reduces the cost of designing different fluorescent probes for each SNP. Therefore, this method can be applied to many biological and genetic studies, such as multiple candidate gene testing, genome‐wide association study, pharmacogenetics, and medical diagnostics.     

Application of fluorescence polarization to enzyme assays and single nucleotide polymorphism genotyping: some recent developments

This article describes some recent developments in the field of fluorescence polarization (FP) as applied to enzyme assays and single nucleotide polymorphism (SNP) genotyping. First, we present our recent progress on the application of fluorescence polarization to high throughput screening (HTS). We show how the use of a 2-thiopyridinone-based, mixed disulfide biotinylation reagent can shorten the assay time of our recently reported kinase assay method based on thiophosphorylation and biotinylation from several hours to a few minutes. We also summarize our recent findings on a new approach for HTS of kinases, proteases and phosphatases based on the use of a cationic poly-amino acid such as polyarginine. We show how the careful selection of the polyarginine concentration and the ionic strength of the solution during the FP measurement allow one to expand the range of substrates that can be assayed. Both of these methods are valuable additions to the existing techniques for HTS. Most importantly, both of these methods can be applied to the assay of kinases without the need for any antibodies. In the area of genomics, we present some results from our studies on a new single nucleotide polymorphism typing approach based on the polymerase catalyzed extension of 3' fluorescein labeled primers. Contrary to our initial expectations, we observed that the enzymatic extension of these primers results in a significant decrease of the fluorescence polarization value. Possible explanations of this phenomenon are discussed.     

Multiplexed SNP genotyping using nanobarcode particle technology

Single-nucleotide polymorphisms (SNP) are the most common form of sequence variation in the human genome. Large-scale studies demand high-throughput SNP genotyping platforms. Here we demonstrate the potential of encoded nanowires for use in a particles-based universal array for high-throughput SNP genotyping. The particles are encoded sub-micron metallic nanorods manufactured by electroplating inert metals such as gold and silver into templates and releasing the resulting striped nanoparticles. The power of this technology is that the particles are intrinsically encoded by virtue of the different reflectivity of adjacent metal stripes, enabling the generation of many thousands of unique encoded substrates. Using SNP found within the cytochrome P450 gene family, and a universal short oligonucleotide ligation strategy, we have demonstrated the simultaneous genotyping of 15 SNP; a format requiring discrimination of 30 encoded nanowires (one per allele). To demonstrate applicability to real-world applications, 160 genotypes were determined from multiplex PCR products from 20 genomic DNA samples.     

A multiplex single nucleotide polymorphism genotyping method using ligase‐based mismatch discrimination and CE‐SSCP

Accuracy, simplicity, and cost‐effectiveness are the most important criteria for a genotyping method for SNPs compatible with clinical use. One method developed for SNP genotyping, ligase‐based discrimination, is considered the simplest for clinical diagnosis. However, multiplex assays using this method are limited by the detection method. Although CE has been introduced as an alternative to error prone microarray‐based detection, the design process and multiplex assay procedure are complicated because of the DNA size‐dependent separation principle. In this study, we developed a simple and accurate multiplex genotyping method using reaction condition‐optimized ligation and high‐resolution CE‐based SSCP. With this high‐resolution CE‐SSCP system, we are able to use similar‐sized probes, thereby eliminating the complex probe design step and simplifying the optimization process. We found that this method could accurately discriminate single‐base mismatches in SNPs of the tp53 gene, used as targets for multiplex detection.     

Highly multiplex and sensitive SNP genotyping method using a three‐color fluorescence‐labeled ligase detection reaction coupled with conformation‐sensitive CE

For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost‐effectiveness are the most important criteria. Among the methods currently being developed for SNP genotyping technology, the ligation‐dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error‐prone hybridization‐based detection, the multiplex assay process is complicated because of the size‐based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high‐resolution CE‐SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar‐sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three‐color fluorescence‐labeled probes.     

Developing a new nonbinary SNP fluorescent multiplex detection system for forensic application in China

Nonbinary single‐nucleotide polymorphisms (SNPs) are potential forensic genetic markers because their discrimination power is greater than that of normal binary SNPs, and that they can detect highly degraded samples. We previously developed a nonbinary SNP multiplex typing assay. In this study, we selected additional 20 nonbinary SNPs from the NCBI SNP database and verified them through pyrosequencing. These 20 nonbinary SNPs were analyzed using the fluorescent‐labeled SNaPshot multiplex SNP typing method. The allele frequencies and genetic parameters of these 20 nonbinary SNPs were determined among 314 unrelated individuals from Han populations from China. The total power of discrimination was 0.9999999999994, and the cumulative probability of exclusion was 0.9986. Moreover, the result of the combination of this 20 nonbinary SNP assay with the 20 nonbinary SNP assay we previously developed demonstrated that the cumulative probability of exclusion of the 40 nonbinary SNPs was 0.999991 and that no significant linkage disequilibrium was observed in all 40 nonbinary SNPs. Thus, we concluded that this new system consisting of new 20 nonbinary SNPs could provide highly informative polymorphic data which would be further used in forensic application and would serve as a potentially valuable supplement to forensic DNA analysis.     

PCR-free MDR1 polymorphism identification by gold nanoparticle probes

Single nucleotide polymorphisms (SNPs) represent the most abundant source of genetic variation in the human genome, and they can be linked to genetic susceptibilities or varied pharmaceutical responses. Established SNP detection techniques are mainly PCR-based, which means that they involve complex, labor-intensive procedures, are easy contaminated, and can give false-positive results. Therefore, we have developed a simple and rapid MS-based disulfide barcode methodology that relies on magnifying the signal from a dual-modified gold nanoparticle. This approach permits direct SNP genotyping of total human genomic DNA without the need for primer-mediated enzymatic amplification. Disulfides that are attached to the gold nanoparticle serve as a “barcode” that allows different sequences to be discerned using MS detection. Specificity is based on two sequential oligonucleotide hybridizations, which include two steps: the first is the capture of the target by gene-specific probes immobilized onto magnetic beads; the second is the recognition of gold nanoparticles functionalized with allele-specific oligonucleotides. The sensitivity of this new method reaches down to the 0.1 fM range, thus approaching that of PCR. The feasability of this SNP identification methodology based on an MS-based disulfide barcode assay was demonstrated by applying it to genomic DNA samples representing all possible genotypes of the SNPs G2677T and C3435T in the human MDR1 gene. Due to its great advantage—the ability to perform SNP typing without the use of PCR—the assay was found to be simple, rapid and robust, and so may be highly suited to routine clinical detection as well as basic medical research.     

Exploring of tri-allelic SNPs using pyrosequencing and the SNaPshot methods for forensic application

Tri-allelic single nucleotide polymorphisms (SNPs) are potential forensic markers for DNA analysis. Currently, only a limited number of tri-allelic SNP loci have been proved to be fit for forensic application. In this study, we aimed to develop an effective method to select and genotype tri-allelic SNPs based on both Pyrosequencing (PSQ) and the SNaPshot methods. 50 candidate SNPs were chosen from NCBI's dbSNP database and were analyzed by PSQ. The results revealed that 20 SNPs were tri-allelic and were located on 16 autosomal chromosomes. Then 20 SNP loci were combined in one multiplex polymerase chain reaction to develop a single base extension (SBE)-based SNP-typing assay. A total of 100 unrelated Chinese individuals were genotyped by this assay and allele frequencies were estimated. The total discrimination power was 0.999999999975 and the cumulative probability of exclusion was 0.9937. These data demonstrated that the strategy is a rapid and effective method for seeking and typing tri-allelic SNPs. In addition, the 20 tri-allelic SNP multiplex typing assay may be used to supplement paternity testing and human identification.     

一种基于磁性纳米粒子PCR的高通量SNP分型方法

利用磁性纳米粒子PCR扩增(MNPs-PCR)和等位基因特异性双色荧光探针(Cy3, Cy5)杂交, 建立了一种单核苷酸多态性(SNP)分型的新方法. 应用该方法对9个样本MTHFR基因的C677T多态进行检测, 野生和突变型样本正错配信号比大于9.0, 杂合型正错配信号比接近1.0, 分型结果经测序验证. 此方法无须产物纯化、浓缩, 扫描分型结果快速、直观, 是一种操作简单、快速、高通量、高灵敏度的分型方法.     

Quadruplex Genotyping of TLR-8 Polymorphisms in a Single Reaction

Toll-like Receptor 8 (TLR8) plays an important role in the innate immune defense against various pathogens. The TLR8 genetic variation affects the course of infections with agents such as HIV, HCV, and mycobacteria. To assess the influence of single nucleotide polymorphisms (SNPs) large cohorts need to be studied; thus, rapid and cost-efficient protocols for high-throughput TLR8 genotyping are needed. We designed a single-tube assay for genotyping four SNPs located to the major coding exon of TLR8 using the LightCycler 480 system. The new method is accurate, fast, low-priced, and can be applied for fully automated high-throughput genotyping of TLR8 SNPs.     

A new multiplex for human identification using insertion/deletion polymorphisms

Human identification is usually based on the study of STRs or SNPs depending on the particular characteristics of the investigation. However, other types of genetic variation such as insertion/deletion polymorphisms (indels) have considerable potential in the field of identification, since they can combine the desirable characteristics of both STRs and SNPs. In this study, a set of 38 non‐coding bi‐allelic autosomal indels reported to be polymorphic in African, European, and Asian populations were selected. We developed a sensitive genotyping assay, which is able to characterize all 38 bi‐allelic markers using a single multiplex PCR and detected with standard CE analyzers. Amplicon length was designed to be shorter than 160 bp. Complete profiles were obtained using 0.3 ng of DNA, and full genotyping of degraded samples was possible in cases where standard STR typing had partially failed. A total of 306 individuals from Angola, Mozambique, Portugal, Macau, and Taiwan were studied and population data are presented. All indels were polymorphic in the three population groups studied and the random match probabilities of the set ranged in orders of magnitude from 10^?14 to 10^?15. Therefore, the indel‐plex represents a valuable approach in human identification studies, especially in challenging DNA cases, as a more straightforward and efficient alternative to SNP typing.     

Integrated platform for detection of DNA sequence variants using capillary array electrophoresis

We have developed a highly versatile platform that performs temperature gradient capillary electrophoresis (TGCE) for mutation/single-nucleotide polymorphism (SNP) detection, sequencing and mutation/SNP genotyping for identification of sequence variants on an automated 24-, 96- or 192-capillary array instrument. In the first mode, multiple DNA samples consisting of homoduplexes and heteroduplexes are separated by CE, during which a temperature gradient is applied that covers all possible temperatures of 50% melting equilibrium (Tms) for the samples. The differences in Tms result in separation of homoduplexes from heteroduplexes, thereby identifying the presence of DNA variants. The sequencing mode is then used to determine the exact location of the mutation/SNPs in the DNA variants. The first two modes allow the rapid identification of variants from the screening of a large number of samples. Only the variants need to be sequenced. The third mode utilizes multiplexed single-base extensions (SBEs) to survey mutations and SNPs at the known sites of DNA sequence. The TGCE approach combined with sequencing and SBE is fast and cost-effective for high-throughput mutation/SNP detection.     

Homogeneous DNA-detection based on the non-enzymatic reactions promoted by target DNA.

Much effort has focused on methods for detecting various genetic differences in individuals, including single nucleotide polymorphisms (SNPs). SNP can be characterized as a substitution, insertion, or deletion at a single base position on a DNA strand. There is expected to be on average one SNP for every 1000 bases of the human genome, and some variations located in genes are suspected to alter both the protein structure and the expression level. Therefore, highly sensitive techniques with a simple procedure would be desirable for a high-throughput screening of millions of SNPs widely dispersed throughout the human genome. In this short review, we consider recently reported unique techniques for genotyping in a homogeneous solution, and organize them in terms of the chemical and physical processes accelerated on DNA.     

Nucleic acid and SNP detection via template‐directed native chemical ligation and inductively coupled plasma mass spectrometry

Detection of nucleic acids and single nucleotide polymorphisms (SNPs) is of pivotal importance in biology and medicine. Given that the biological effect of SNPs often is enhanced in combination with other SNPs, multiplexed SNP detection is desirable. We show proof of concept of the multiplexed detection of SNPs based on the template‐directed native chemical ligation (NCL) of PNA‐probes carrying a metal tag allowing detection using ICP‐MS. For the detection of ssDNA oligonucleotides (30 bases), two probes, one carrying the metal tag and a second one carrying biotin for purification, are covalently ligated. The methodological limit of detection is of 29 pM with RSD of 6.7% at 50 pM (n = 5). Detection of SNPs is performed with the combination of two sets of reporter probes. The first probe set targets the SNP, and its yield is compared with a second set of probes targeting a neighboring sequence. The assay was used to simultaneously differentiate between alleles of three SNPs at 5‐nM concentration.