A novel MALDI-TOF based methodology for genotyping single nucleotide polymorphisms

A new MALDI-TOF based detection assay was developed for analysis of single nucleotide polymorphisms (SNPs). It is a significant modification on the classic three-step minisequencing method, which includes a polymerase chain reaction (PCR), removal of excess nucleotides and primers, followed by primer extension in the presence of dideoxynucleotides using modified thermostable DNA polymerase. The key feature of this novel assay is reliance upon deoxynucleotide mixes, lacking one of the nucleotides at the polymorphic position. During primer extension in the presence of depleted nucleotide mixes, standard thermostable DNA polymerases dissociate from the template at positions requiring a depleted nucleotide; this principal was harnessed to create a genotyping assay. The assay design requires a primer- extension primer having its 3'-end one nucleotide upstream from the interrogated site. The assay further utilizes the same DNA polymerase in both PCR and the primer extension step. This not only simplifies the assay but also greatly reduces the cost per genotype compared to minisequencing methodology. We demonstrate accurate genotyping using this methodology for two SNPs run in both singleplex and duplex reactions. We term this assay nucleotide depletion genotyping (NUDGE). Nucleotide depletion genotyping could be extended to other genotyping assays based on primer extension such as detection by gel or capillary electrophoresis.     

MagSNiPer: a new single nucleotide polymorphism typing method based on single base extension, magnetic separation, and chemiluminescence

We have developed a new method for typing single nucleotide polymorphisms (SNPs), MagSNiPer, based on single base extension, magnetic separation, and chemiluminescence. Single base nucleotide extension reaction is performed with a biotinylated primer whose 3' terminus is contiguous to the SNP site with a tag-labeled ddNTP. Then the primers are captured by magnetic-coated beads with streptavidin, and unincorporated labeled ddNTP is removed by magnetic separation. The magnetic beads are incubated with anti-tag antibody conjugated with alkaline phosphatase. After the removal of excess conjugates by magnetic separation, SNP typing is performed by measuring chemiluminescence. The incorporation of labeled ddNTP is monitored by chemiluminescence induced by alkaline phosphatase. MagSNiPer is a simple and robust SNP typing method with a wide dynamic range and high sensitivity. Using MagSNiPer, we could perform SNP typing with as little as 10(-17) mol of template DNA.     

Software for machine-independent quantitative interpretation of SSCP in capillary array electrophoresis (QUISCA)

PCR single-stranded conformational polymorphism (SSCP) analysis is a simple and rapid electrophoretic technique for the sensitive detection of sequence variants of PCR products. Here we describe a cross-platform program package, quantitative interpretation of SSCP in capillary array (QUISCA), which allows semi-automated quantitative detection of sequence variants separated by multicolor fluorescence-based SSCP electrophoresis using various capillary array apparatus. The program, together with the QUISCAview as a graphical user interface, takes trace data in ASCII format and processes them with three modules: signal denoising/baseline subtraction, color-matrix construction/application, and calibration of peak positions between multiple capillary runs using internal standard peaks. QUISCA is compatible with data from various widely used capillary array sequencers and is suitable not only for finding or typing SNPs in individual DNAs but also for the accurate estimation of the allele frequencies of many SNPs using a pooled DNA strategy. QUISCA can also serve as a versatile core program for various fragment analyses.     

A versatile fluorescence-based multiplexing assay for CAPS genotyping on capillary electrophoresis systems

Recent advances in next-generation sequencing techniques and the development of genomics resources for crop plants with large genomes allow the detection of a large number of single nucleotide polymorphisms (SNPs) and their use in a high-throughput manner. However, such large numbers of SNPs are on the one hand not needed in some plant breeding projects and on the other hand not affordable in some cases, raising the need for fast and low-cost innovative techniques for marker detection. In marker selection in plant breeding programs, cleaved amplified polymorphic sequence (CAPS) markers still play a significant role as a complement to other high-throughput methods for SNP genotyping. New methods focusing on the acceleration of CAPS-based genotyping are therefore highly desirable. The combination of the classical CAPS method and a M13-tailed primer multiplexing assay was used to develop an agarose-gel-free protocol for the analysis of SNPs via restriction enzyme digestion. PCR products were fluorescence-labeled with a universal M13 primer and subsequently digested with the appropriate restriction endonuclease. After mixing differently labeled products, they were detected in a capillary electrophoresis system. This method allowed the cost-effective genotyping of several SNPs in barley in a multiplexed manner at an overall low cost in a short period of time. This new method was efficiently combined with the simultaneous detection of simple sequence repeats in the same electrophoresis run, resulting in a procedure well suited for marker-based selection procedures, genotyping of mapping populations and the assay of genetic diversity.     

Efficient validation of single nucleotide polymorphisms in plants by allele-specific PCR,with an example from barley

Although the increasing number of expressed sequence tags (ESTs) from the public domain has facilitated the detection of single nucleotide polymorphisms (SNPs), further validation is needed before they can be used as markers. For SNP validation, we have compared 2 independent methods: (1) the primer extension method followed by capillary electrophoresis on an ABI PRISM 3100 Genetic Analyzer and (2) nested PCR followed by agarose-based visualization. We present an assessment of the efficiency and costs associated with these methods, based on a sample of barley cultivars.     

单核苷酸多态性检测方法研究进展

：单核苷酸多态性（singlenucleotidepolymorphism,SNP）是指在基因组水平上由单个核苷酸的变异引起的一种DNA序列多态性。SNP作为第三代分子标记,具有数量多、分布广等特点,已成为人类后基因组时代的主要研究内容之一。单核苷酸多态性在医学研究、临床诊断、药物开发与合理用药、法医学、遗传学的发展方面具有重要意义。因此,建立高度自动化和高通量的SNP检测分析技术十分重要。各种SNP分型检测方法都由等位基因特异性的识别反应和等住基因识别产物的分析检测两个部分组成。本文系统的介绍了引物延伸反应、序列杂交反应、酶连接反应、酶切割反应、核酸链构象差异反应等SNP检测的等位基因特异性的识别原理,以及质谱、荧光共振和偏振信号、化学发光、毛细管电泳测序、生物传感器等分析检测手段,并简要介绍了相关识别原理和分析检测手段的优缺点及应用范围,并对SNP检测技术的发展进行了展望。     

Analysis of genetic variation in the GenomEUtwin project.

Multiallelic short tandem repeat polymorphisms, or microsatellites, are useful markers in genome wide scans to identify chromosomal regions containing genes underlying disease loci. The biallelic single nucleotide polymorphism (SNP) can be used to fine map previously identified large candidate regions or to test functional candidate genes by association analysis. In the GenomEUtwin project the population based impact of susceptibility genes for six multifactorial traits will be studied. A genome wide panel of informative human microsatellite markers will be analyzed by fluorescent capillary electrophoresis in well characterized twin and population samples. Contrary to microsatellites, selection of the most informative panels of SNPs is hampered by imperfect data on the allele frequencies and population distribution of SNPs markers in the databases. Therefore, selection of SNPs requires a substantial amount of bioinformatics, and, the SNPs need to be validated experimentally in the relevant populations prior to genotyping large sample sets. In the GenomEUtwin project, large scale genotyping of SNPs will be performed using the SNPstreamUHT and MassARRAY genotyping systems that are based on the primer extension reaction principle combined with fluorescent and mass spectrometric detection, respectively. Production of the genotyping data will be a joint effort by GenomEUtwin partners at the University of Helsinki, the National Public Health Institute in Helsinki, Finland and Uppsala University, Sweden. All genotyping data will be stored in a common database established specifically for the GenomEUtwin project, from where it can be accessed by the twin research centres that provided the samples for genotyping.     

Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides

Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled oligonucleotide primers was mixed with biotin-ddNTPs, DNA polymerase and the DNA templates containing the SNPs in a single tube. The nucleotide at the 3′-end of each CFET-labeled oligonucleotide primer was complementary to a particular SNP in the template. Only the CFET-labeled primer that is fully complementary to the DNA template was extended by DNA polymerase with a biotin-ddNTP. We isolated the DNA extension fragments that carry a biotin at the 3′-end by capture with streptavidin-coated magnetic beads, while the unextended primers were eliminated. The biotinylated fluorescent DNA fragments were subsequently analyzed in a multicolor fluorescence electrophoresis system. The distinct fluorescence signature and electrophoretic mobility of each DNA extension product in the electropherogram coded the SNPs without the use of a sizing standard. We simultaneously distinguished six nucleotide variations in synthetic DNA templates and a PCR product from the retinoblastoma tumor suppressor gene. The use of CFET-labeled primers and biotin-ddNTPs coupled with the specificity of DNA polymerase in SBE offered a multiplex method for detecting SNPs.     

Multiplex automated primer extension analysis: simultaneous genotyping of several polymorphisms.

Accurate and fast genotyping of single nucleotide polymorphisms (SNPs) is of significant scientific importance for linkage and association studies. We report here an automated fluorescent method we call multiplex automated primer extension analysis (MAPA) that can accurately genotype multiple known SNPs simultaneously. This is achieved by substantially improving a commercially available protocol (SNaPshot). This protocol relies on the extension of a primer that ends one nucleotide 5'of a given SNP with fluorescent dideoxy-NTPs (minisequencing), followed by analysis on an ABI PRisMS 377 Semi-Automated DNA Sequencer Our modification works by multiplexing the initial reaction that produces the DNA template for primer extension and/or multiplexing several primers (corresponding to several SNPs) in the same primer extension reaction. Then, we run each multiplexed reaction on a single gel lane. We demonstrate that MAPA can be used to genotype up to four SNPs simultaneously, even in compound heterozygote samples, with complete accuracy (based on concordance with sequencing results). We also show that primer design, unlike the DNA template purification method, can significantly affect genotyping accuracy, and we suggest useful guidelines for quick optimization.     

Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays

Detection of DNA sequence variation is critical to biomedical applications, including disease genetic identification, diagnosis and treatment, drug discovery and forensic analysis. Here, we describe an arrayed primer extension-based genotyping method (APEX-2) that allows multiplex (640-plex) DNA amplification and detection of single nucleotide polymorphisms (SNPs) and mutations on microarrays via four-color single-base primer extension. The founding principle of APEX-2 multiplex PCR requires two oligonucleotides per SNP/mutation to generate amplicons containing the position of interest. The same oligonucleotides are then subsequently used as immobilized single-base extension primers on a microarray. The method described here is ideal for SNP or mutation detection analysis, molecular diagnostics and forensic analysis. This robust genetic test has minimal requirements: two primers, two spots on the microarray and a low cost four-color detection system for the targeted site; and provides an advantageous alternative to high-density platforms and low-density detection systems.     

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.     

Competitive enzymatic reaction to control allele-specific extensions

Here, we present a novel method for SNP genotyping based on protease-mediated allele-specific primer extension (PrASE), where the two allele-specific extension primers only differ in their 3′-positions. As reported previously [Ahmadian,A., Gharizadeh,B., O'Meara,D., Odeberg,J. and Lundeberg,J. (2001), Nucleic Acids Res., 29, e121], the kinetics of perfectly matched primer extension is faster than mismatched primer extension. In this study, we have utilized this difference in kinetics by adding protease, a protein-degrading enzyme, to discriminate between the extension reactions. The competition between the polymerase activity and the enzymatic degradation yields extension of the perfectly matched primer, while the slower extension of mismatched primer is eliminated. To allow multiplex and simultaneous detection of the investigated single nucleotide polymorphisms (SNPs), each extension primer was given a unique signature tag sequence on its 5′ end, complementary to a tag on a generic array. A multiplex nested PCR with 13 SNPs was performed in a total of 36 individuals and their alleles were scored. To demonstrate the improvements in scoring SNPs by PrASE, we also genotyped the individuals without inclusion of protease in the extension. We conclude that the developed assay is highly allele-specific, with excellent multiplex SNP capabilities.     

Fluorescence resonance energy transfer (FRET) using ssDNA binding fluorescent dye

There is a need for simple and inexpensive methods for genotyping single nucleotide polymorphisms (SNPs) and short insertion/deletion variations (InDels). In this work, I demonstrate that a single-stranded DNA (ssDNA) binding dye can be used as a donor fluorophore for fluorescence resonance energy transfer (FRET). The method presented is a homogenous assay in which detection is based on the FRET from the fluorescence of the ssDNA dye bound to the unmodified detection primer to the fluorescent nucleotide analog incorporated into this detection primer during cyclic template directed primer extension reaction. Collection of the FRET emission spectrum with a scanning fluorescence spectrophotometer allows powerful data analysis. The fluorescence emission signal is modified by the optical properties of the assay vessel. This seems to be a completely neglected parameter. By proper selection of the optical properties of the assay plate one can improve the detection of the fluorescence emission signal.     

Fluorescence-based single-strand conformation polymorphism analysis of the low density lipoprotein receptor gene by capillary electrophoresis

We describe here a new method to screen for unknown mutations in the low density lipoprotein (LDL) receptor gene by the use of capillary electrophoresis in single-strand conformation polymorphism (SSCP) analysis. To analyze the promoter and all 18 exons, 20 different amplification reactions were necessary. For each polymerase chain reaction (PCR), the forward and reverse primers were 5′ fluorescent-labelled with FAM and HEX, respectively. To test the accuracy of the newly developed method, 61 genetic variants distributed in 16 exons were analyzed. Under identical electrophoresis conditions (13 kV, 30°C, 30 min), 59 mutations were detected by a distinct abnormal SSCP pattern. The two remaining mutations showed only slight abnormalities, which could be amplified by increasing the electrophoresis temperature. The high accuracy, the degree of automation and the speed of analysis make fluorescence-based SSCP analysis with capillary electrophoresis ideal for rapid mutation screening and the technique is well-suited for clinical applications.     

Characterization of sequence polymorphisms from microsatellite flanking regions in <Emphasis Type="Italic">Vitis</Emphasis> spp

Single nucleotide polymorphisms or SNPs are the most abundant form of genetic variation in the genome of plants and animals. Microsatellites are hypervariable regions of genome, while their flanking regions are assumed to be as conserved as the average of the genome. In the present study, flanking sequences of 10 microsatellite loci were compared in different cultivars of Vitis to determine the existing polymorphism. For every microsatellite, about 8 homozygous cultivars (regarding the microsatellite genotype) were chosen for sequencing. A total of 45 different varieties of Vitis and 91 sequences were analysed. Sequence polymorphisms were detected for all the microsatellite flanking regions studied, including single nucleotide polymorphisms (SNPs), insertions and deletions. The number of identified changes varied considerably among the loci with a frequency of one polymorphism every 41 nucleotides, being VVMD5 the most polymorphic one. A number of SNPs were used to design SNP markers, which were scored by dideoxy single base primer extension and capillary electrophoresis methodology. These SNP markers were employed to genotype 21 cultivars of Vitis vinifera and 4 varieties of other Vitis species. The utility of the markers developed as well as their utility for varietal identification and pedigree studies is discussed, using a similar study carried out with the 10 microsatellites as a reference.     

Single-nucleotide polymorphisms detected in expressed sequence tags of melon (Cucumis melo L.).

A search was performed for single-nucleotide polymorphisms (SNP) and short insertions-deletions (indels) in 34 melon (Cucumis melo L.) expressed sequence tag (EST) fragments between two distantly related melon genotypes, a group Inodorus 'Piel de sapo' market class breeding line T111 and the Korean accession PI 161375. In total, we studied 15 kb of melon sequence. The average frequency of SNPs between the two genotypes was one every 441 bp. One indel was also found every 1666 bp. Seventy-five percent of the polymorphisms were located in introns and the 3'untranslated regions. On average, there were 1.26 SNPs plus indels per amplicon. We explored three different SNP detection systems to position five of the SNPs in a melon genetic map. Three of the SNPs were mapped using cleaved amplified polymorphic sequence (CAPS) markers, one SNP was mapped using the single primer extension reaction with fluorescent-labelled dideoxynucleotides, and one indel was mapped using polyacrilamide gel electrophoresis separation. The discovery of SNPs based on ESTs and a suitable system for SNP detection has broad potential utility in melon genome mapping.     

Use of bovine EST data and human genomic sequences to map 100 gene-specific bovine markers

A system to use bovine EST data in conjunction with human genomic sequence to improve the bovine linkage map over the entire genome or on specific chromosomes was evaluated. Bovine EST sequence was used to provide primer sequences corresponding to bovine genes, while human genomic sequence directed primer design to flank introns and produce amplicons of appropriate size for efficient direct sequencing. The sequence tagged sites (STS) produced in this way from the four sires of the MARC reference families were examined for single nucleotide polymorphisms (SNPs) that could be used to map the corresponding genes. With this approach, along with a primer/extension mass spectrometry SNP genotyping assay, 100 ESTs were placed on the bovine genetic linkage map. The first 70 were chosen at random from bovine EST–human genomic comparisons. An additional 30 ESTs were successfully mapped to bovine Chromosome 19 (BTA19), and comparison of the resulting BTA19 map to the position of the corresponding human orthologs on the HSA17 draft sequences revealed differences in the spacing and order of genes. Over 80% of successful amplicons contained SNPs, indicating that this is an efficient approach to generating EST-associated genetic markers. We have demonstrated the feasibility of constructing a linkage map based on SNPs associated with ESTs and the plausibility of utilizing EST, comparative mapping information, and human sequence data to target regions of the bovine genome for SNP marker development.     

Fluorescence-based,high-throughput DNA polymerase assay

The commonly used DNA polymerase assay is based on the detection of incorporated radiolabeled nucleotides in a DNA elongation reaction. It is laborious, radioactive, and can be highly variable. Here we report a nonradioactive fluorescence-based assay. The method consists of Cydye-labeled nucleotides, biotinylated primer, and a streptavidin-coated microplate. The assay is found to have sensitivity and dynamic range comparable to the classical radioactive method. Moreover, it has the advantages of being simple, stable, nonradioactive, and suitable for high-throughput applications. We have also found that, to ensure efficient measurement of the enzyme activity, the template DNA used in this method should have a sequence that avoids the incorporation of the fluorescence-labeled nucleotide in a consecutive way.     

Approach to analysis of single nucleotide polymorphisms by automated constant denaturant capillary electrophoresis

Melting gel techniques have proven to be amenable and powerful tools in point mutation and single nucleotide polymorphism (SNP) analysis. With the introduction of commercially available capillary electrophoresis instruments, a partly automated platform for denaturant capillary electrophoresis with potential for routine screening of selected target sequences has been established. The aim of this article is to demonstrate the use of automated constant denaturant capillary electrophoresis (ACDCE) in single nucleotide polymorphism analysis of various target sequences. Optimal analysis conditions for different single nucleotide polymorphisms on ACDCE are evaluated with the Poland algorithm. Laboratory procedures include only PCR and electrophoresis. For direct genotyping of individual SNPs, the samples are analyzed with an internal standard and the alleles are identified by co-migration of sample and standard peaks.In conclusion, SNPs suitable for melting gel analysis based on theoretical thermodynamics were separated by ACDCE under appropriate conditions. With this instrumentation (ABI 310 Genetic Analyzer), 48 samples could be analyzed without any intervention. Several institutions have capillary instrumentation in-house, thus making this SNP analysis method accessible to large groups of researchers without any need for instrument modification.     

MC1R是控制鸡黑色素形成的候选主效基因

黑素皮质素受体1 (melanocortin 1-receptor, MC1R)基因是控制动物黑色素合成的重要基因.采用多聚酶链反应-单链构象多态性分析(PCR-SSCP)以及DNA测序的方法,在由丝羽乌骨鸡与明星肉鸡为亲本建立的中国农业大学资源家系群体鸡MC1R基因的编码区检测到3个单核苷酸多态位点,并对该单核苷酸多态性进行了分析.结果显示,鸡MC1R基因编码区引物3扩增片段多态性是由G→A（867位）点突变引起的,引物5扩增片段的多态性是由C→T（1 292位）与C→G（1 377位）两个点突变引起的,最后对单核苷酸多态性与肤色、肉色、胫色与内脏膜色等黑色素性状进行了卡方独立性分析,结果显示,MC1R基因编码区867处突变与鸡的肤色性状显著相关（P＜0.05）,1 292处突变与鸡的活体胫色性状显著相关（P＜0.05）,1 377处突变与鸡的肉色性状显著相关（P＜0.05）.研究表明,MC1R基因可能是鸡黑色素性状的主效基因或者与鸡控制黑色素性状的主效基因连锁.