Rapid method for detecting SNPs on agarose gels and its application in candidate gene mapping

TILLING (Targeting Induced Local Lesions IN Genomes) exploits the fact that CEL I endonuclease cleaves heteroduplexes at positions of single nucleotide or small indel mismatches. To detect single nucleotide polymorphisms (SNPs) across a population, DNA pools are created and a target locus under query is PCR-amplified and subjected to heteroduplex formation, followed by CEL I cleavage. Currently, the common method used to detect cleaved products is by polyacrylamide gel electrophoresis using a high-throughput genotyping platform. Exact SNPs are then determined by sequencing. We sought to simplify the detection of CEL I-cleaved products on conventional agarose gels to make the technique more accessible to collaborating partners in developing countries where access to instrumentation could be limiting. Here, we used a panel of stress-related genes to evaluate SNP detection across 48 rice genotypes by contrasting them individually against IR64 and Nipponbare. SNP detection calls corresponded perfectly with those obtained from the Li-Cor genotypers. We were able to detect SNPs in pools of eight DNA templates, suggesting that the agarose gel system could be used to screen for SNPs with comparable throughput as that of the Li-Cor genotypers and showed that the throughput can be increased by analyzing larger amplicons (∼3 kb). The agarose method offers a significant advantage by alleviating the need for labeled primers. We further demonstrated that the agarose method can be effectively used in gene mapping, an application particularly useful for parental lines with low levels of polymorphism. The lower cost and simplicity of the technique make it possible for broader applications of SNP-based markers for germplasm characterization and mapping studies.     

Multiplex genotyping of the human beta2-adrenergic receptor gene using solid-phase capturable dideoxynucleotides and mass spectrometry

Previously, we established the feasibility of using solid phase capturable (SPC) dideoxynucleotides to generate single base extension (SBE) products which were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for multiplex genotyping, an approach that we refer to as SPC-SBE. We report here the expanding of the SPC-SBE method as a single-tube assay to simultaneously detect 20 single nucleotide variations in a model system and 3 single nucleotide polymorphisms (SNPs) in the human beta2-adrenergic receptor (beta2AR) gene. Twenty primers were designed to have a sufficient mass difference between all extension products for accurate detection of nucleotide variants of the synthetic templates related to the p53 gene. These primers were extended simultaneously in a single tube with biotin-ddNTPs to generate 3(')-biotinylated DNA products, which were first captured by streptavidin-coated magnetic beads and then released from the beads and analyzed with MALDI-TOF MS. This approach generates a mass spectrum free of primer peaks and their associated dimers, increasing the scope of multiplexing SNPs. We also simultaneously genotyped 3 SNPs in the beta2AR gene (5(')LC-Cys19Arg, Gly16Arg, and Gln27Glu) from the genomic DNA of 20 individuals. Comparison of this approach with direct sequencing and the restriction fragment length polymorphism method indicated that the SPC-SBE method is superior for detecting nucleotide variations at known SNP sites.     

Specific detection of bacillus anthracis using a TaqMan mismatch amplification mutation assay

Single nucleotide polymorphisms (SNPs) are increasingly recognized as important diagnostic markers for the detection and differentiation of Bacillus anthracis. The use of SNP markers for identifying B. anthracis DNA in environmental samples containing genetically similar bacteria requires the ability to amplify and detect DNA with single nucleotide specificity. We designed a TaqMan mismatch amplification mutation assay (TaqMAMA) around a SNP in the plcR gene of B. anthracis. The assay permits specific, low-level detection (25 fg DNA) of this B. anthracis-specific SNP, even in the presence of environmental DNA extracts containing a 20,000-fold excess of the alternate allele. We anticipate that the ability to selectively amplify and detect low copy number DNAs with single nucleotide specificity will represent a valuable tool in the arena of biodefense and microbial forensics.     

Estimation of single nucleotide polymorphism allele frequency in DNA pools by using Pyrosequencing

Positional cloning of genes underlying complex diseases, such as type 2 diabetes mellitus (T2DM), typically follows a two-tiered process in which a chromosomal region is first identified by genome-wide linkage scanning, followed by association analyses using densely spaced single nucleotide polymorphic markers to identify the causal variant(s). The success of genome-wide single nucleotide polymorphism (SNP) detection has resulted in a vast number of potential markers available for use in the construction of such dense SNP maps. However, the cost of genotyping large numbers of SNPs in appropriately sized samples is nearly prohibitive. We have explored pooled DNA genotyping as a means of identifying differences in allele frequency between pools of individuals with T2DM and unaffected controls by using Pyrosequencing technology. We found that allele frequencies in pooled DNA were strongly correlated with those in individuals (r=0.99, P<0.0001) across a wide range of allele frequencies (0.02-0.50). We further investigated the sensitivity of this method to detect allele frequency differences between contrived pools, also over a wide range of allele frequencies. We found that Pyrosequencing was able to detect an allele frequency difference of less than 2% between pools, indicating that this method may be sensitive enough for use in association studies involving complex diseases where a small difference in allele frequency between cases and controls is expected.     

A single multiplex PCR and SNaPshot minisequencing reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups

SNaPshot minisequencing reaction is in increasing use because of its fast detection of many polymorphisms in a single assay. In this work we described a highly sensitive single nucleotide polymorphisms (SNPs) typing method with detection of 42 mitochondrial DNA (mtDNA) SNPs in a single PCR and SNaPshot multiplex reaction, in order to allow haplogroup classification in Latin American admixture population. We validated the panel typing 160 Brazilian individuals. Complete SNP profiles were obtained from 10 pg of total DNA. We conclude that it is possible to build and genotype more than forty mtDNA SNPs in a single multiplex PCR and SNaPshot reaction, with sensitivity and reliability, resolving haplogroup classification in admixture populations.     

Detection of single nucleotide polymorphisms within the Listeria genus using an 'asymmetric' fluorogenic probe set and fluorescence resonance energy transfer based-PCR

AIMS: We describe a novel and inexpensive fluorescence energy transfer (FRET)-based PCR protocol to distinguish single nucleotide polymorphisms (SNPs) within the genus Listeria. METHODS AND RESULTS: Sequence information for the 16S rRNA gene of representative Listeria species was used to design genus-specific primers and two species-specific probes that differed in sequence by one single nucleotide. The probes were 5' labelled with either fluorescein or Texas Red, quenched with a shorter yet complementary 3' dimethyl-amino-phenyloazo benzoic acid (DABCYL) labelled oligonucleotide, and then incorporated into a previously reported 'asymmetric' FRET-based PCR detection protocol. CONCLUSIONS: Listeria monocytogenes could be readily distinguished from other members of the Listeria genus after PCR amplification and measurement of endpoint fluorescence at two different wavelengths. SIGNIFICANCE AND IMPACT OF THE STUDY: The relatively low cost and high flexibility of this system will benefit laboratories in their efforts to develop rapid and specific methods to detect minor sequence differences between related microorganisms.     

Dimer of 2,7-diamino-1,8-naphthyridine for the detection of mismatches formed by pyrimidine nucleotide bases

Discrimination of base mismatches from normal Watson-Crick base pairs in duplex DNA constitutes a key approach to the detection of single nucleotide polymorphisms (SNPs). We have developed a sensor for a surface plasmon resonance (SPR) assay system to detect G-G, A-A, and C-C mismatch duplexes by employing a surface upon which mismatch-binding ligands (MBLs) are immobilized. We synthesized a new MBL consisting of 2,7-diamino-1,8-naphthyridine (damND) and immobilized it onto a CM5 sensor chip to carry out the SPR assay of DNA duplexes containing a single-base mismatch. The SPR sensor with damND revealed strong responses to all C-C mismatches, and sequence-dependent C-T and T-T mismatches. Compared to ND- and naphthyridine-azaquinolone hybrid (NA)-immobilized sensor surfaces, with affinity to mismatches composed of purine nucleotide bases, the damND-immobilized surface was useful for the detection of the mismatches composed of pyrimidine nucleotide bases.     

Olive variety identification by ligation detection reaction in a universal array format

Molecular methodologies are increasingly being developed to assess the origin and authenticity of raw organic materials and processed food products. Here we describe the application of a microarray-based assay for single nucleotide polymorphisms (SNPs) identification in olive cultivars. The assay distinguishes alleles in a ligation detection reaction (LDR), with subsequent fluorescent detection by hybridization on a universal array (UA). The LDR-UA approach was used to detect 17 SNPs in olive genomic sequences previously amplified by PCR from fresh olive leaves. Genotype calls obtained with the LDR-UA were in full agreement with those determined by direct sequencing. The panel of 17 SNPs is sufficient to discriminate 49 olive varieties selected among the most widely cultivated for olive oil production in the Mediterranean area.     

Number of SNPS loci needed to detect population structure

The study of the association of polymorphic genetic markers with common diseases is one of the most powerful tools in modern genetics. Interest in single nucleotide polymorphisms (SNPs) has steadily grown over the last decade. SNPs are currently the most developed markers in the human genome because they have a number of advantages over other marker types. One of the critical problems responsible for 'spurious' association findings in case-control studies is population stratification. There are many statistical approaches developed for detecting population heterogeneity. However the power to detect population structure by known methods is highly dependent on the number of loci utilised. We performed an analysis of SNPs data available in the public domain from The Single Nucleotide Consortia Ltd. (TSCL). Three populations, Afro-American, Asian and Caucasian, were compared. Estimation of the minimum number of SNPs loci necessary for detection of the population structure was performed. Two clustering approaches, distance-based and model-based, were compared. The model-based approach was superior when compared with the distance-based method. We found more than 65 random SNPs loci are required for identifying distinct geographically separated populations. Increasing the number of markers to over 100 raises the probability of correct assignment of a particular individual to an origin group to over 90%, even with conventional clustering methods.     

A modified mutation detection method for large-scale cloning of the possible single nucleotide polymorphism sequences

Although the human genome has been nearly completely sequenced, the functions and the roles of the vast majority of the genes, and the influences of single nucleotide polymorphisms (SNPs) in these genes are not entirely known. A modified mutation detection method was developed for large-scale cloning of the possible SNPs between tumor and normal cells for facilitating the identification of genetic factors that associated with cancer formation and progression. The method involves hybridization of restriction enzyme-cut chromosomal DNA, cleavage and modification of the sites of differences by enzymes, and differential cloning of sequence variations with a designed vector. Experimental validations of the presence and location of sequence variations in the isolated clones by PCR and DNA sequencing support the capability of this method in identifying sequence differences between tumor cells and normal cells.     

Fast identification of biological pathways associated with a quantitative trait using group lasso with overlaps

Where causal SNPs (single nucleotide polymorphisms) tend to accumulate within biological pathways, the incorporation of prior pathways information into a statistical model is expected to increase the power to detect true associations in a genetic association study. Most existing pathways-based methods rely on marginal SNP statistics and do not fully exploit the dependence patterns among SNPs within pathways.We use a sparse regression model, with SNPs grouped into pathways, to identify causal pathways associated with a quantitative trait. Notable features of our "pathways group lasso with adaptive weights" (P-GLAW) algorithm include the incorporation of all pathways in a single regression model, an adaptive pathway weighting procedure that accounts for factors biasing pathway selection, and the use of a bootstrap sampling procedure for the ranking of important pathways. P-GLAW takes account of the presence of overlapping pathways and uses a novel combination of techniques to optimise model estimation, making it fast to run, even on whole genome datasets.In a comparison study with an alternative pathways method based on univariate SNP statistics, our method demonstrates high sensitivity and specificity for the detection of important pathways, showing the greatest relative gains in performance where marginal SNP effect sizes are small.     

Automated identification of single nucleotide polymorphisms from sequencing data

The single nucleotide polymorphism (SNP) is the difference of the DNA sequence between individuals and provides abundant information about genetic variation. Large scale discovery of high frequency SNPs is being undertaken using various methods. However, the publicly available SNP data sometimes need to be verified. If only a particular gene locus is concerned, locus-specific polymerase chain reaction amplification may be useful. Problem of this method is that the secondary peak has to be measured. We have analyzed trace data from conventional sequencing equipment and found an applicable rule to discern SNPs from noise. The rule is applied to multiply aligned sequences with a trace and the peak height of the traces are compared between samples. We have developed software that integrates this function to automatically identify SNPs. The software works accurately for high quality sequences and also can detect SNPs in low quality sequences. Further, it can determine allele frequency, display this information as a bar graph and assign corresponding nucleotide combinations. It is also designed for a person to verify and edit sequences easily on the screen. It is very useful for identifying de novo SNPs in a DNA fragment of interest.     

Nucleic acid probe containing fluorescent tricyclic base-linked acyclonucleoside for detection of single nucleotide polymorphisms

The development of a reliable and simple method for detecting single nucleotide polymorphisms (SNPs), common genetic variations in the human genome, is currently an important research area because SNPs are important for identifying disease-causing genes and for pharmacogenetic studies. Here, we developed a novel method for SNP detection. We designed and synthesized DNA probes containing a fluorescent tricyclic base-linked acyclonucleoside P. The type of nucleobases involved in the SNP sites in the DNA and RNA targets could be determined using four DNA probes containing P. Thus, this system would provide a novel and simple method for detecting SNPs in DNA and RNA targets.     

Screening and scanning of single nucleotide polymorphisms in the pig melanocortin 1 receptor gene (MC1R) by pyrosequencing.

The increasing interest in the discovery and characterization of single nucleotide polymorphisms (SNPs) emphasis the need for high-throughput and cost effective scoring methods. Pyrosequencing is a novel method for screening SNPs. In this study we examine breed specific SNPs in the pig melanocortin 1 receptor gene (MC1R), some causing coat color phenotypes. A total of fifteen pigs representing eight breeds and crosses were analyzed by pyrosequencing. In addition to nine previously known SNPs, we also detected one new missense mutation by pyrosequencing. We here show that the SNPs were readily scored using standard reaction conditions. Insertions as well as substitutions were unambiguously detected and all genotypes were resolved in terms of homo- and heterozygozity.     

Selecting tagging SNPs for association studies using power calculations from genotype data

Recent studies have indicated that linkage disequilibrium (LD) between single nucleotide polymorphism (SNP) markers can be used to derive a reduced set of tagging SNPs (tSNPs) for genetic association studies. Previous strategies for identifying tSNPs have focused on LD measures or haplotype diversity, but the statistical power to detect disease-associated variants using tSNPs in genetic studies has not been fully characterized. We propose a new approach of selecting tSNPs based on determining the set of SNPs with the highest power to detect association. Two-locus genotype frequencies are used in the power calculations. To show utility, we applied this power method to a large number of SNPs that had been genotyped in Caucasian samples. We demonstrate that a significant reduction in genotyping efforts can be achieved although the reduction depends on genotypic relative risk, inheritance mode and the prevalence of disease in the human population. The tSNP sets identified by our method are remarkably robust to changes in the disease model when small relative risk and additive mode of inheritance are employed. We have also evaluated the ability of the method to detect unidentified SNPs. Our findings have important implications in applying tSNPs from different data sources in association studies.     

单核苷酸多态性检测分析技术

单核苷酸多态性(SNP)作为第三代遗传标记已经广泛用于基因作图、疾病相关性分析、群体遗传学及药物研究等领域。 文中系统地介绍了目前国内外主要的SNP检测技术,任何一种SNP的检测方法都可将之看成由两部分组成,即区分SNP位点的原理方法和数据的检测分析手段,文章对这两部分做了较详细的介绍,并对SNP检测技术的发展进行了展望。 Abstract :As the third generation of genetic markers SNPs（single nucleotide polymorphisms）has been used extentively in gene mapping,disease-correlativity analysis ,population genetics and drug research.Here methods for detection are reviewed.Most SNP genotyping are a combination of method for interrogating SNPs and analysis tecnique.It described both parts and give a outlook for detection.     

Estimate of nucleotide diversity in dogs with a pool-and-sequence method

Nucleotide diversity (π), the average number of base differences per site for two homologous sequences randomly selected from a population, is an important parameter used to understand the structure and history of populations. It is also important for determining the feasibility of developing a genetic map for a species from single nucleotide polymorphisms (SNPs). Nucleotide diversity has never been estimated for dogs. Segments of twelve canine genes from ten diverse dog breeds were examined for nucleotide variation by using a pool-and-sequence method. We identified three SNPs in the coding regions (2501 bp) and 11 SNPs in the introns (2953 bp). Each of these putative SNPs was tested by restriction enzyme analysis, and all were verified. Six additional SNPs were identified in a single SINE contained in one gene. Using these data, canine sequence diversity across breeds was estimated to be 0.001 and 0.0004 in intronic and coding regions, respectively, with SNPs spaced every 400 bp on average. Discovery of useful SNPs in 7 of the 12 genes suggests that construction of a canine SNP-based map can be accomplished with current technology. Thirteen polymorphic SNPs were also found in 5847 bp in the cat, horse, ox, and pig, by using four of the same genes from which canine nucleotide diversity was estimated. These results suggest that these species may have similar amounts of nucleotide diversity. Received: 1 February 2000 / Accepted: 22 August 2000     

DNA microarrays on a dendron-modified surface improve significantly the detection of single nucleotide variations in the p53 gene

Selectivity and sensitivity in the detection of single nucleotide polymorphisms (SNPs) are among most important attributes to determine the performance of DNA microarrays. We previously reported the generation of a novel mesospaced surface prepared by applying dendron molecules on the solid surface. DNA microarrays that were fabricated on the dendron-modified surface exhibited outstanding performance for the detection of single nucleotide variation in the synthetic oligonucleotide DNA. DNA microarrays on the dendron-modified surface were subjected to the detection of single nucleotide variations in the exons 5–8 of the p53 gene in genomic DNAs from cancer cell lines. DNA microarrays on the dendron-modified surface clearly discriminated single nucleotide variations in hotspot codons with high selectivity and sensitivity. The ratio between the fluorescence intensity of perfectly matched duplexes and that of single nucleotide mismatched duplexes was >5–100 without sacrificing signal intensity. Our results showed that the outstanding performance of DNA microarrays fabricated on the dendron-modified surface is strongly related to novel properties of the dendron molecule, which has the conical structure allowing mesospacing between the capture probes. Our microarrays on the dendron-modified surface can reduce the steric hindrance not only between the solid surface and target DNA, but also among immobilized capture probes enabling the hybridization process on the surface to be very effective. Our DNA microarrays on the dendron-modified surface could be applied to various analyses that require accurate detection of SNPs.