Characterization of genomic single nucleotide polymorphism via colorimetric detection using a single gold nanoprobe

Identification of specific nucleic acid sequences mediated by gold nanoparticles derivatized thiol-modified oligonucleotides (Au–nanoprobes) has been proven to be a useful tool in molecular diagnostics. Here, we demonstrate that, on optimization, detection may be simplified via the use of a single Au–nanoprobe to detect a single nucleotide polymorphism (SNP) in homo- or heterozygote condition. We validated this non-cross-linking approach through the analysis of 20 clinical samples using a single specific Au–nanoprobe for an SNP in the FTO (fat mass and obesity-associated) gene against direct DNA sequencing. Sensitivity, specificity, and limit of detection (LOD) were determined, and statistical differences were calculated by one-way analysis of variance (ANOVA) and a post hoc Tukey’s test to ascertain whether there were any differences between Au–nanoprobe genotyped groups. For the first time, we show that the use of a single Au–nanoprobe can detect SNP for each genetic status (wild type, heterozygous, or mutant) with high degrees of sensitivity (87.50%) and specificity (91.67%).     

Genotyping of single nucleotide polymorphism using model-based clustering

MOTIVATION: Single nucleotide polymorphisms have been investigated as biological markers and the representative high-throughput genotyping method is a combination of the Invader assay and a statistical clustering method. A typical statistical clustering method is the k-means method, but it often fails because of the lack of flexibility. An alternative fast and reliable method is therefore desirable. RESULTS: This paper proposes a model-based clustering method using a normal mixture model and a well-conceived penalized likelihood. The proposed method can judge unclear genotypings to be re-examined and also work well even when the number of clusters is unknown. Some results are illustrated and then satisfactory genotypings are shown. Even when the conventional maximum likelihood method and the typical k-means clustering method failed, the proposed method succeeded.     

Usefulness of single nucleotide polymorphism data for estimating population parameters

Single nucleotide polymorphism (SNP) data can be used for parameter estimation via maximum likelihood methods as long as the way in which the SNPs were determined is known, so that an appropriate likelihood formula can be constructed. We present such likelihoods for several sampling methods. As a test of these approaches, we consider use of SNPs to estimate the parameter Theta = 4N(e)micro (the scaled product of effective population size and per-site mutation rate), which is related to the branch lengths of the reconstructed genealogy. With infinite amounts of data, ML models using SNP data are expected to produce consistent estimates of Theta. With finite amounts of data the estimates are accurate when Theta is high, but tend to be biased upward when Theta is low. If recombination is present and not allowed for in the analysis, the results are additionally biased upward, but this effect can be removed by incorporating recombination into the analysis. SNPs defined as sites that are polymorphic in the actual sample under consideration (sample SNPs) are somewhat more accurate for estimation of Theta than SNPs defined by their polymorphism in a panel chosen from the same population (panel SNPs). Misrepresenting panel SNPs as sample SNPs leads to large errors in the maximum likelihood estimate of Theta. Researchers collecting SNPs should collect and preserve information about the method of ascertainment so that the data can be accurately analyzed.     

Ligation detection reaction-TaqMan procedure for single nucleotide polymorphism detection on genomic DNA

In this article, we describe a genotyping approach applicable to both individual and multiplexed single nucleotide polymorphism (SNP) analysis, based on a ligation detection reaction (LDR) performed directly on genomic DNA. During the ligation, the biallelic state of the SNP locus is converted into a bimarker state of ligated detector oligonucleotides. The state of the markers is then determined by a 5'-nuclease assay (TaqMan) with universal fluorescent probes. The LDR-TaqMan method was successfully applied for the genotyping of 30 SNP loci of Arabidopsis thaliana. The technology is cost-effective, needs no locus-specific optimization, requires minimal manipulations, and has very good potential for automation.     

Multiplex real-time single nucleotide polymorphism detection and quantification by quencher extension

Multiplex quencher extension (multiplex-QEXT) is a novel closed tube single-step method for detection and quantification of several single nucleotide polymorphisms (SNPs) simultaneously. The principle of multiplex-QEXT is that 5' reporter-labeled probes are 3' single-base-extended with TAMRA dideoxy nucleotides if the respective SNP alleles are present. TAMRA can serve as either an energy acceptor (quencher-based detection) or donor [fluorescence resonance energy transfer (FRET)-based detection] for a wide range of different reporter fluorochromes. The extension can therefore be recorded by the respective reporter fluorescence change. We evaluated multiplex-QEXT, analyzing four different SNP loci in the Listeria monocytogenes inlA gene. Probes labeled with the reporters 6-FAM, TET, VIC, and Alexa Fluor 594 were used. Responses for the fluorochromes 6-FAM, TET, and VIC were detected by quenching (decreased fluorescence), while the response for Alexa Fluor 594 was detected by FRET (increased fluorescence). We evaluated the SNP-allele pattern in 252 different L. monocytogenes strains. Multiplex-QEXT gave a good resolution, detecting seven major and five minor groups of L. monocytogenes. Comparison with serotyping showed that multiplex-QEXT gave better resolution. We also evaluated the quantitative aspects of multiplex-QEXT. Quantitative information was obtained for all the fluorochrome/probe combinations in the sample pools. The detection limits for 6-FAM, TET and Alexa Fluor 594 were the presence of the 10% target SNP alleles (P < 0.05), while the detection limit for VIC was the presence of the 5% target SNP alleles (P < 0.05). Currently, overlap in the fluorescence emission spectra is the limiting factor for the multiplexing potential of QEXT. With the emergence of new fluorochromes with narrow emission spectra, we foresee great potential for increasing the multiplex level in the future.     

Efficient and cost-effective single nucleotide polymorphism detection with different fluorescent applications.

Three methods-5'nuclease assay with TaqMan, minisequencing, and oligonucleotide ligation assay (OLA)-were compared to detectfive single nucleotide polymorphisms (SNPs) in three separate genes. Each method had advantages and disadvantages. The 5' nuclease assay was the fastest and required only a single step. OLA was the most time consuming to optimize, but once running it was the least expensive method. Minisequencing was universal; however, the technique was also the most expensive. All three methods were reliable and highly effective. Investigators must consider their goals in terms of time, sample number, and expense when selecting among these genotyping techniques.     

Detection of single nucleotide mutations in wheat using single strand conformation polymorphism gels

We show that single strand conformation polymorphism (SSCP) analysis, using the mutation detection enhancement (MDE^TM) matrix, is efficient at detecting sequence polymorphisms in PCR amplicons. Four independent wheat genomic fragments were amplified from two contrasting templates and sequenced. The allelic fragments were differentiated at 1–6 single nucleotide positions, but MDE-SSCP was able to unequivocally distinguish each allelic pair. The approach is therefore considered a powerful way of identifying single nucleotide polymorphisms (SNPs) without extensive amplicon sequencing.     

A new method for detecting single nucleotide polymorphism using GFP-display

The single nucleotide polymorphism (SNP) of aldehyde dehydrogenase-2 (ALDH2) codon 487, GAA (Glu) or AAA (Lys), was examined using green fluorescent protein (GFP)-display, an electrophoretic detection method for single amino acid changes. Although no shift in migration between the GFP-ALDH (Glu487) and GFP-ALDH (Lys487) fusion proteins was observed on SDS/urea gel, the two migrated to different positions when tagged with Asp. The SNP analysis was performed with GFP-ALDH-Asp3, and GFP-ALDH-Asp3 constructed from donors having the codon GAA/GAA, GAA/AAA or AAA/AAA was detected as different patterns as expected. GFP-display is potentially a unique method in SNP analysis, which does not require any special equipment or chemicals.     

Development of single nucleotide polymorphism markers in the large and complex rubber tree genome using next-generation sequence data

The development of single nucleotide polymorphism (SNP) markers provides the opportunity to improve many areas of plant breeding and population genetics. Unfortunately, for species such as the rubber tree (Hevea brasiliensis), the use of next-generation sequencing for genomic SNP discovery is very difficult because of the large genome size and the abundance of repeated sequences. Access to a set of validated SNP markers is a significant advantage for rubber researchers who wish to apply SNPs in scientific research. Here, we performed genomic sequencing of H. brasiliensis and generated 10,993,648 short reads, which were assembled into 10,071 contigs (N50 = 3078) by a de novo assembly strategy. A total of 2446 contigs presented no hits in the current H. brasiliensis genome assembly and may therefore be considered novel genomic sequences of rubber tree. A total of 143 putative polymorphic positions were selected, gene annotations were available for 58.7 % of the markers, and all of the sequences could be anchored to the released H. brasiliensis genome. These SNPs were validated in eight genotypes of H. brasiliensis and 15 F1 plants from a mapping population, resulting in 30 (20.9 %) positions correctly classified. The analysis revealed key candidate genes responsible for defence mechanisms and provided markers for further genetic improvement of Hevea in breeding programmes.     

Oligonucleotide ligation assay-based DNA chip for multiplex detection of single nucleotide polymorphism

An oligonucleotide ligation assay-based DNA chip has been developed to detect single nucleotide polymorphism. Synthesized nonamers, complementary to the flanking sequences of the mutation sites in target DNA, were immobilized onto glass slides through disulfide bonds on their 5' terminus. Allele-specific pentamers annealed adjacent to the nonamers on the complementary target DNA, containing 5'-phosphate groups and biotin labeled 3'-ends, were mixed with the target DNA in tube. Ligation reactions between nonamers and pentamers were carried out on chips in the presence of T4 DNA ligase. Ligation products were directly visualized on chips through enzyme-linked assay. The effect of G:T mismatch at different positions of pentamers on the ligation were evaluated. The results showed that any mismatch between pentamer and the target DNA could lead to the decrease of ligation, which can be detected easily. The established approach was further used for multiplex detection of mutations in rpoB gene of rifampin-resistant Mycobacterium tuberculosis clinical isolates.     

SNPHunter: a bioinformatic software for single nucleotide polymorphism data acquisition and management

Background  

Single nucleotide polymorphisms (SNPs) provide an important tool in pinpointing susceptibility genes for complex diseases and in unveiling human molecular evolution. Selection and retrieval of an optimal SNP set from publicly available databases have emerged as the foremost bottlenecks in designing large-scale linkage disequilibrium studies, particularly in case-control settings.     

RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

Background

The combinatorial library strategy of using multiple candidate ligands in mixtures as library members is ideal in terms of cost and efficiency, but needs special screening methods to estimate the affinities of candidate ligands in such mixtures. Herein, a new method to screen candidate ligands present in unknown molar quantities in mixtures was investigated.

Results

The proposed method involves preparing a processed-mixture-for-screening (PMFS) with each mixture sample and an exogenous reference ligand, initiating competitive binding among ligands from the PMFS to a target immobilized on magnetic particles, recovering target-ligand complexes in equilibrium by magnetic force, extracting and concentrating bound ligands, and analyzing ligands in the PMFS and the concentrated extract by chromatography. The relative affinity of each candidate ligand to its reference ligand is estimated via an approximation equation assuming (a) the candidate ligand and its reference ligand bind to the same site(s) on the target, (b) their chromatographic peak areas are over five times their intercepts of linear response but within their linear ranges, (c) their binding ratios are below 10%. These prerequisites are met by optimizing primarily the quantity of the target used and the PMFS composition ratio. The new method was tested using the competitive binding of biotin derivatives from mixtures to streptavidin immobilized on magnetic particles as a model. Each mixture sample containing a limited number of candidate biotin derivatives with moderate differences in their molar quantities were prepared via parallel-combinatorial-synthesis (PCS) without purification, or via the pooling of individual compounds. Some purified biotin derivatives were used as reference ligands. This method showed resistance to variations in chromatographic quantification sensitivity and concentration ratios; optimized conditions to validate the approximation equation could be applied to different mixture samples. Relative affinities of candidate biotin derivatives with unknown molar quantities in each mixture sample were consistent with those estimated by a homogenous method using their purified counterparts as samples.

Conclusions

This new method is robust and effective for each mixture possessing a limited number of candidate ligands whose molar quantities have moderate differences, and its integration with PCS has promise to routinely practice the mixture-based library strategy.     

A single nucleotide polymorphism genotyping method using phosphate-affinity polyacrylamide gel electrophoresis

To date, various methods have been developed to facilitate the genotyping of a single nucleotide polymorphism (SNP) for aiding in the diagnosis and treatment of inherited diseases. The most commonly used method for SNP genotyping is an allele-specific hybridization procedure using an expensive fluorochrome-labeled oligonucleotide probe and a specialized fluorescence analyzer. Here, we introduce a simple and reliable genotyping method using a 1:1 mixture of 5'-phosphate-labeled and nonlabeled allele-specific polymerase chain reaction (PCR) primers. The method is based on the difference in mobility of the phosphorylated and nonphosphorylated PCR products (in the same number of basepairs) on phosphate-affinity polyacrylamide gel electrophoresis. The phosphate-affinity site is a polyacrylamide-bound dinuclear zinc(II) complex, which preferentially captures the 5'-phosphate-labeled allele-specific product compared with the corresponding nonlabeled product. The obtained DNA migration bands can be visualized by ethidium bromide staining. We demonstrate the genotyping of a SNP reported in a human cardiac sodium channel gene, SCN5A, using this novel procedure.     

Nanoparticle-based detection and quantification of DNA with single nucleotide polymorphism (SNP) discrimination selectivity

Sequence-specific DNA detection is important in various biomedical applications such as gene expression profiling, disease diagnosis and treatment, drug discovery and forensic analysis. Here we report a gold nanoparticle-based method that allows DNA detection and quantification and is capable of single nucleotide polymorphism (SNP) discrimination. The precise quantification of single-stranded DNA is due to the formation of defined nanoparticle-DNA conjugate groupings in the presence of target/linker DNA. Conjugate groupings were characterized and quantified by gel electrophoresis. A linear correlation between the amount of target DNA and conjugate groupings was found. For SNP detection, single base mismatch discrimination was achieved for both the end- and center-base mismatch. The method described here may be useful for the development of a simple and quantitative DNA detection assay.     

Simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography

We report here a novel method to simultaneously detect CpG methylation and single nucleotide polymorphisms (SNPs) using denaturing high performance liquid chromatography (DHPLC). PCR products of bisulfite-modified CpG islands were separated using DHPLC. BstUI digestion and DNA sequencing were used in confirmation studies. Consistent with the BstUI digestion assay, the 294 bp PCR product of the modified hMLH1 promoter showed different retention times between the methylated cell lines (RKO and Cla, 6.7 min) and the unmethylated cell lines (PACM82 and MGC803, 6.2 min). No hMLH1 methylation was observed in 13 primary gastric carcinomas and their matched normal tissues. One hMLH1 SNP was detected in gastric cancer patients, in both cancer and normal tissues. DNA sequencing revealed that the SNP is a G→A variation at –93 nt of the hMLH1 promoter. A two-peak chromatogram was also obtained in the 605 bp PCR product of the Cox-2 promoter of the AGS, HEK293 and MKN45 cell lines by DHPLC. Another peak corresponding to methylated CpG islands was observed on the chromatogram of the Cox-2-methylated AGS cell line after bisulfite treatment. In conclusion, methylation in homoallelic and heteroallelic CpG islands could be detected rapidly and reliably by bisulfite–DHPLC. A SNP in the target sequence could also be detected at the same time.     

Fluorescence detection of single nucleotide polymorphisms using a universal molecular beacon

We present a simple and novel assay—employing a universal molecular beacon (MB) in the presence of Hg²⁺—for the detection of single nucleotide polymorphisms (SNPs) based on Hg²⁺–DNA complexes inducing a conformational change in the MB. The MB (T₇-MB) contains a 19-mer loop and a stem of a pair of seven thymidine (T) bases, a carboxyfluorescein (FAM) unit at the 5′-end, and a 4-([4-(dimethylamino)phenyl]azo)benzoic acid (DABCYL) unit at the 3′-end. Upon formation of Hg²⁺–T₇-MB complexes through T–Hg²⁺–T bonding, the conformation of T₇-MB changes from a random coil to a folded structure, leading to a decreased distance between the FAM and DABCYL units and, hence, increased efficiency of fluorescence resonance energy transfer (FRET) between the FAM and DABCYL units, resulting in decreased fluorescence intensity of the MB. In the presence of complementary DNA, double-stranded DNA complexes form (instead of the Hg²⁺–T₇-MB complexes), with FRET between the FAM and DABCYL units occurring to a lesser extent than in the folded structure. Under the optimal conditions (20 nM T₇-MB, 20 mM NaCl, 1.0 μM Hg²⁺, 5.0 mM phosphate buffer solution, pH 7.4), the linear plot of the fluorescence intensity against the concentration of perfectly matched DNA was linear over the range 2–30 nM (R² = 0.991), with a limit of detection of 0.5 nM at a signal-to-noise ratio of 3. This new probe provides higher selectivity toward DNA than that exhibited by conventional MBs.