High-throughput SNP genotyping based on solid-phase PCR on magnetic nanoparticles with dual-color hybridization

Single-nucleotide polymorphisms (SNPs) are one-base variations in DNA sequence that can often be helpful when trying to find genes responsible for inherited diseases. In this paper, a microarray-based method for typing single nucleotide polymorphisms (SNPs) using solid-phase polymerase chain reaction (PCR) on magnetic nanoparticles (MNPs) was developed. One primer with biotin-label was captured by streptavidin coated magnetic nanoparticles (SA-MNPs), and PCR products were directly amplified on the surface of SA-MNPs in a 96-well plate. The samples were interrogated by hybridization with a pair of dual-color probes to determine SNP, and then genotype of each sample can be simultaneously identified by scanning the microarray printed with the denatured fluorescent probes. The C677T polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene from 126 samples were interrogated using this method. The results showed that three different genotypes were discriminated by three fluorescence patterns on the microarray. Without any purification and reduction procedure, and all reactions can be performed in the same vessel, this approach will be a simple and labor-saving method for SNP genotyping and can be applicable towards the automation system to achieve high-throughput SNP detection.     

Allele-specific hybridization using streptavidin-coated magnetic beads for species identification, S genotyping, and SNP analysis in plants

Dot-blot hybridization has been successfully used for the construction of single nucleotide polymorphism (SNP)-based linkage maps, quantitative trait locus analysis, marker-assisted selection, and the identification of species and cultivars. This method is, however, time-consuming, even for a small number of plant samples. We propose a method in which streptavidin-coated magnetic beads replace the nylon membrane for immobilization of the PCR products and are hybridized with allele-specific oligonucleotide probes and a digoxigenin-labeled oligonucleotide hybridized with the allele-specific oligonucleotide probe. After amplification of plant DNA by PCR with the biotinylated primers, those oligonucleotide probes having species-specific or allele-specific sequences were mixed together with the digoxigenin-labeled oligonucleotide and the streptavidin-coated magnetic beads at a temperature suitable for each probe. Species-specific internal transcribed spacer 1 (ITS1) sequences and allele-specific sequences of the hypervariable region I of S-locus receptor kinase (SRK) specifically detected ITS1 sequences and SRK alleles in Brassica species, respectively. SNPs were also successfully analyzed by using allele-specific oligonucleotide probes and competitive oligonucleotides. In the SNP analysis, PCR products were indirectly captured by magnetic beads. SNP alleles of eight cultivars each of Brassica rapa and Raphanus sativus were analyzed using streptavidin-coated magnetic beads. The genotyping results corresponded well with those of dot-blot-SNP analysis. Although allele-specific hybridization using streptavidin-coated magnetic beads is somewhat costly, it is easier and more rapid than dot-blot hybridization. This method is suitable for the analysis of a small number of plant samples with a large number of DNA markers.     

基于磁性颗粒“在位”PCR和通用标签技术的新型高通量SNP分型方法

单核苷酸多态性(single nucleotide polymorphism,SNP)在对复杂疾病遗传易感性以及基于群体基因识别等方面的研究中起着非常重要的作用,尤其是对复杂疾病遗传易感性的研究,需要对大量样本进行分型.为了满足这种要求,亟待需要发展一种操作简单、成本较低、适于自动化和高通量的分型技术.利用磁性颗粒"在位"固相PCR(insituMPs-PCR)扩增的靶序列,通过与野生、突变标签探针以及双色荧光(Cy3,Cy5)通用检测子杂交实现对样本的分型.应用该方法,对96个样本的亚甲基四氢叶酸还原酶(MTHFR)基因C677T位点的多态性进行了检测,其野生型和突变型样本的正错配信号比大于4.5,杂合型正错配信号比接近1,分型结果与测序结果一致.     

PCR amplification on magnetic nanoparticles: application for high-throughput single nucleotide polymorphism genotyping

A novel approach for the genotyping of single nucleotide polymorphisms (SNPs) based on solidphase PCR on magnetic nanoparticles (MNPs) is described. PCR products were amplified directly on MNPs. The genotypes of a given SNP were differentiated by hybridization with a pair of allele-specific probes labeled with dual-color fluorescence (Cy3, Cy5). The results were analyzed by scanning the microarray printed with the denatured fluorescent probes on an unmodified glass slide. Electrophoresis analysis indicated that PCR could proceed successfully when MNPs-bound primers were used. Furthermore, nine different samples were genotyped and their fluorescent signals were quantified. Genotyping results showed that three genotypes for the locus were very easily discriminated. The fluorescent ratios (match probe:mismatch probe signal) of homozygous samples were over 9.3, whereas heterozygous samples had ratios near 1.0. Without any purification and concentration of PCR products, this new MNP-PCR based genotyping assay potentially provides a rapid, labor-saving method for genotyping of a large number of individuals.     

近交系小鼠双色荧光正相杂交芯片技术体系的建立和优化

目的探讨采用单核苷酸多态性（SNP）检测方法-双色荧光正相杂交芯片技术对近交系小鼠遗传质量监测及相关影响因素。方法运用基于芯片的双色荧光正相杂交检测SNP技术,进行芯片杂交动力学研究,考察信号值（Cy3,Cy5）和ratio值（Cy5/Cy3）与PCR产物点样浓度、PCR产物长度和荧光标记探针长度之间的关系,研究PCR产物点样浓度、PCR产物长度和荧光标记探针长度对SNP分型的影响。结果采用正反标记实验后,Ratio值随着PCR产物点样浓度的增加呈稳定趋势;PCR双链产物长度对信号值影响比较大,点样时其长度不宜太长,最好不超过450 bp;随荧光标记探针长度的增加,基因分型能力明显下降,长度为15 bp最佳,长度超过20 bp时,已基本没有区分能力。结论PCR产物点样浓度、PCR产物长度和荧光标记探针长度是双色荧光正相杂交SNP分型系统的重要影响因素,采取适当的PCR产物点样浓度、PCR产物长度和荧光标记探针长度,并采用正反标记实验,可以取得稳定、准确的基因分型效果。为进一步进行近交系小鼠遗传质量监测的研究奠定基础。     

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.     

Single nucleotide polymorphism detection in aldehyde dehydrogenase 2 (ALDH2) gene using bacterial magnetic particles based on dissociation curve analysis

Single nucleotide polymorphism (SNP) detection for aldehyde dehydrogenase 2 (ALDH2) gene based on DNA thermal dissociation curve analysis was successfully demonstrated using an automated system with bacterial magnetic particles (BMPs) by developing a new method for avoiding light scattering caused by nanometer-size particles when using commercially available fluorescent dyes such as FITC, Cy3, and Cy5 as labeling chromophores. Biotin-labeled PCR products in ALDH2, two allele-specific probes (Cy3-labeled detection probe for ALDH2*1 and Cy5-labeled detection probe for ALDH2*2), streptavidin-immobilized BMPs (SA-BMPs) were simultaneously mixed. The mixture was denatured at 70 degrees C for 3 min, cooled slowly to 25 degrees C, and incubated for 10 min, allowing the DNA duplex to form between Cy3- or Cy5-labeled detection probes and biotin-labeled PCR products on SA-BMPs. Then duplex DNA-BMP complex was heated to 58 degrees C, a temperature determined by dissociation curve analysis and a dissociated single-base mismatched detection probe was removed at the same temperature under precise control. Furthermore, fluorescence signal from the detection probe was liberated into the supernatant from completely matched duplex DNA-BMP complex by heating to 80 degrees C and measured. In the homozygote target DNA (ALDH2*1/*1 and ALDH2*2/*2), the fluorescence signals from single-base mismatched were decreased to background level, indicating that mismatched hybridization was efficiently removed by the washing process. In the heterozygote target DNA (ALDH2*1/*2), each fluorescence signals was at a similar level. Therefore, three genotypes of SNP in ALDH2 gene were detected using the automated detection system with BMPs.     

Rapid high resolution single nucleotide polymorphism-comparative genome hybridization mapping in Caenorhabditis elegans

We have developed a significantly improved and simplified method for high-resolution mapping of phenotypic traits in Caenorhabditis elegans using a combination of single nucleotide polymorphisms (SNPs) and oligo array comparative genome hybridization (array CGH). We designed a custom oligonucleotide array using a subset of confirmed SNPs between the canonical wild-type Bristol strain N2 and the Hawaiian isolate CB4856, populated with densely overlapping 50-mer probes corresponding to both N2 and CB4856 SNP sequences. Using this method a mutation can be mapped to a resolution of ~200 kb in a single genetic cross. Six mutations representing each of the C. elegans chromosomes were detected unambiguously and at high resolution using genomic DNA from populations derived from as few as 100 homozygous mutant segregants of mutant N2/CB4856 heterozygotes. Our method completely dispenses with the PCR, restriction digest, and gel analysis of standard SNP mapping and should be easy to extend to any organism with interbreeding strains. This method will be particularly powerful when applied to difficult or hard-to-map low-penetrance phenotypes. It should also be possible to map polygenic traits using this method.     

Copy number variations identified in the chicken using a 60K SNP BeadChip

Copy number variation (CNV) is considered an important genetic variation, contributing to many economically important traits in the chicken. Although CNVs can be detected using a comparative genomic hybridization array, the high‐density SNP array has provided an alternative way to identify CNVs in the chicken. In the current study, a chicken 60K SNP BeadChip was used to identify CNVs in two distinct chicken genetic lines (White Leghorn and dwarf) using the penncnv program. A total of 209 CNV regions were identified, distributing on chromosomes 1–22 and 24–28 and encompassing 13.55 Mb (1.42%) of chicken autosomal genome area. Three of seven selected CNVs (73.2% individuals) were completely validated by quantitative PCR. To our knowledge, this is the first report in the chicken identifying CNVs using a SNP array. Identification of 190 new identified CNVs illustrates the feasibility of the chicken 60K SNP BeadChip to detect CNVs in the chicken, which lays a solid foundation for future analyses of associations of CNVs with economically important phenotypes in chickens.     

Single nucleotide polymorphism genotyping of aldehyde dehydrogenase 2 gene using a single bacterial magnetic particle

A single nucleotide polymorphism (SNP) genotyping for aldehyde dehydrogenase 2 gene (ALDH2) has been developed by using a nano-sized magnetic particle, which was synthesized intracellularly by magnetic bacteria. Streptavidin-immobilized on bacterial magnetic particles (BMPs) were prepared using biotin labeled cross-linkers reacting with the amine group on BMPs. ALDH2 fragments from genomic DNA were amplified using a TRITC labeled primer and biotin labeled primer pair, and conjugated onto BMP surface by biotin-streptavidin interaction. PCR product-BMP complex was observed at a single particle level by fluorescence microscopy. These complexes were treated with restriction enzyme, specifically digesting the wild-type sequence of ALDH2 (normal allele of ALDH2). The homozygous (ALDH2*1/*1), heterozygous (ALDH2*1/*2), and mutant (ALDH2*2/*2) genotypes were discriminated by three fluorescence patterns of each particle. SNP genotyping of ALDH2 has been successfully achieved at a single particle level using BMP.     

Improvement of the dot-blot-SNP technique for efficient and cost-effective genotyping

Although the dot-blot-SNP technique is a laborsaving, cost-effective method for SNP genotyping of a large number of plants, the synthesis of 5′-digoxigenin (DIG)-labeled oligonucleotides for use as probes is still costly. We developed two probe-labeling methods for this technique, one being digoxigenin labeling of oligonucleotides by PCR (PCR-DIG labeling) and the other being hybridization using a bridge probe and a 5′-DIG-labeled oligonucleotide (bridge hybridization). Bridge hybridization detected allele-specific signals under hybridization conditions similar to those for the 5′-DIG-labeled oligonucleotides and biotin-labeled oligonucleotides, while signals were detected only under a lower stringency condition by PCR-DIG labeling. As a method for genotyping using many markers at one time, two methods, i.e., PCR using mixed primer pairs and hybridization using mixed probes, were examined with successful results. Eighty-five SNP markers designed for genotyping of rice cultivars detected allele-specific signals, the genotyping results corresponding to the previously reported ones.     

Calibration-curve-free quantitative PCR: a quantitative method for specific nucleic acid sequences without using calibration curves

We have developed a simple quantitative method for specific nucleic acid sequences without using calibration curves. This method is based on the combined use of competitive polymerase chain reaction (PCR) and fluorescence quenching. We amplified a gene of interest (target) from DNA samples and an internal standard (competitor) with a sequence-specific fluorescent probe using PCR and measured the fluorescence intensities before and after PCR. The fluorescence of the probe is quenched on hybridization with the target by guanine bases, whereas the fluorescence is not quenched on hybridization with the competitor. Therefore, quench rate (i.e., fluorescence intensity after PCR divided by fluorescence intensity before PCR) is always proportional to the ratio of the target to the competitor. Consequently, we can calculate the ratio from quench rate without using a calibration curve and then calculate the initial copy number of the target from the ratio and the initial copy number of the competitor. We successfully quantified the copy number of a recombinant DNA of genetically modified (GM) soybean and estimated the GM soybean contents. This method will be particularly useful for rapid field tests of the specific gene contamination in samples.     

Simple SNP typing assay using a base-discriminating fluorescent probe

We have developed a new concept involving a single-step homogeneous method for single-nucleotide polymorphism (SNP) typing. In this method, a probe containing base-discriminating fluorescent (BDF) bases is added to a sample solution. BDF base-containing DNA usually shows only a weak fluorescence, but emits a strong blue fluorescence when it recognizes a target base at a specific site in a hybridized strand. By utilizing this feature, a simple mix-and-read SNP typing assay was achieved without any tedious probe-designing or washing processes for exclusion of hybridization error or any addition of DNA-modifying enzymes. This is very different from conventional methods. We simultaneously analyzed a number of samples with ease, with a high accuracy, using our BDF assay.     

双色荧光杂交芯片在检测 P 1A 1M P I 基C 因多态性中的应用

目的：应用一种新的高通量SNP检测方法-双色荧光杂交芯片技术检测CYPIA1 MspI基因多态性。方法：收集江苏汉族人群原发性肺癌患者75例和相应对照77例,应用双色荧光杂交芯片技术检测了152例样本的CYPIAI基因MspI基因多态性,并应用PCR-RFLP技术验证双色荧光杂交芯片的特异性。结果：152例样本的CYPIAI基因双色荧光杂交芯片技术分型结果与PCR-RFLP结果完全相符,两种方法的基因型分型结果具有很好的一致性。结论：双色荧光杂交芯片技术是一个高通量SNP检测的良好工具,特异性高,在大规模人群SNP筛检中具有良好的发展前案。     

Loss-of-heterozygosity analysis of small-cell lung carcinomas using single-nucleotide polymorphism arrays

Human cancers arise by a combination of discrete mutations and chromosomal alterations. Loss of heterozygosity (LOH) of chromosomal regions bearing mutated tumor suppressor genes is a key event in the evolution of epithelial and mesenchymal tumors. Global patterns of LOH can be understood through allelotyping of tumors with polymorphic genetic markers. Simple sequence length polymorphisms (SSLPs, or microsatellites) are reliable genetic markers for studying LOH, but only a modest number of SSLPs are used in LOH studies because the genotyping procedure is rather tedious. Here, we report the use of a highly parallel approach to genotype large numbers of single-nucleotide polymorphisms (SNPs) for LOH, in which samples are genotyped for nearly 1,500 loci by performing 24 polymerase chain reactions (PCR), pooling the resulting amplification products and hybridizing the mixture to a high-density oligonucleotide array. We characterize the results of LOH analyses on human small-cell lung cancer (SCLC) and control DNA samples by hybridization. We show that the patterns of LOH are consistent with those obtained by analysis with both SSLPs and comparative genomic hybridization (CGH), whereas amplifications rarely are detected by the SNP array. The results validate the use of SNP array hybridization for tumor studies.     

Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports.

A simple and rapid method for the analysis of genetic polymorphisms has been developed using allele-specific oligonucleotide arrays bound to glass supports. Allele-specific oligonucleotides are covalently immobilized on glass slides in arrays of 3 mm spots. Genomic DNA is amplified by PCR using one fluorescently tagged primer oligonucleotide and one biotinylated primer oligonucleotide. The two complementary DNA strands are separated, the fluorescently tagged strand is hybridized to the support-bound oligonucleotide array, and the hybridization pattern is detected by fluorescence scanning. Multiple polymorphisms present in the PCR product may be detected in parallel. The effect of spacer length, surface density and hybridization conditions were evaluated, as was the relative efficacy of hybridization with single or double-stranded PCR products. The utility of the method was demonstrated in the parallel analysis of 5 point mutations from exon 4 of the human tyrosinase gene.     

Development of the Handy Bio-Strand and its application to genotyping of OPRM1 (A118G)

We previously developed a three-dimensional microarray system, the Bio-Strand, which exhibits advantages in automated DNA analysis in combination with our Magtration Technology. In the current study, we have developed a compact system for the Bio-Strand, the Handy Bio-Strand, which consists of several tools for the preparation of Bio-Strand Tip, hybridization, and detection. Using the Handy Bio-Strand, we performed single nucleotide polymorphism (SNP) genotyping of OPRM1 (A118G) by allele-specific oligonucleotide competitive hybridization (ASOCH). DNA fragments containing SNP sites were amplified from genomic DNA by PCR and then were fixed on a microporous nylon thread. Thus, prepared Bio-Strand Tip was hybridized with allele-specific Cy5 probes (<15mer), on which the SNP site was designed to be located in the center. By optimizing the amount of competitors, the selectivity of Cy5 probes increased without a drastic signal decrease. OPRM1 (A118G) genotypes of 23 human genomes prepared from whole blood samples were determined by ASOCH using the Handy Bio-Strand. The results were perfectly consistent with those determined by PCR direct sequencing. ASOCH using the Handy Bio-Strand would be a very simple and reliable method for SNP genotyping for small laboratories and hospitals.     

DNA-probes for the highly sensitive identification of single nucleotide polymorphism using single-molecule spectroscopy

This article presents a new, highly sensitive method for the identification of single nucleotide polymorphisms (SNPs) in homogeneous solutions using fluorescently labeled hairpin-structured oligonucleotides (smart probes) and fluorescence single-molecule spectroscopy. While the hairpin probe is closed, fluorescence intensity is quenched due to close contact between the chromophore and several guanosine residues. Upon hybridization to the respective target SNP sequence, contact is lost and the fluorescence intensity increases significantly. High specificity is achieved by blocking sequences containing mismatch with unlabeled oligonucleotides. Time-resolved single-molecule fluorescence spectroscopy enables the detection of individual smart probes passing a small detection volume. This method leads to a subnanomolar sensitivity for this single nucleotide specific DNA assay technique.     

Multiplex single nucleotide polymorphisms genotyping using solid-phase single base extension on magnetic nanoparticles

To fulfill the increasing need for large-scale genetic research, we have developed a new solid-phase single base extension (SBE) protocol on magnetic nanoparticles (MNPs) for multiplex SNP detection using adapter polymerase chain reaction (PCR) products as templates. Extension primers were covalently immobilized on the MNPs, and allele-specific extension took place along the stretch of target DNA for one-color ddNTP incorporation. The MNPs with fluorophores were spotted on a glass slide to fabricate a “bead array” to discriminate their genotypes. Eight SNP loci of three DNA samples were interrogated, and the experiment demonstrated that it is an efficient method for large-scale SNP genotyping.     

In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens

A sensitive and selective genomagnetic assay for the electrochemical detection of food pathogens based on in situ DNA amplification with magnetic primers has been designed. The performance of the genomagnetic assay was firstly demonstrated for a DNA synthetic target by its double-hybridization with both a digoxigenin probe and a biotinylated capture probe, and further binding to streptavidin-modified magnetic beads. The DNA sandwiched target bound on the magnetic beads is then separated by using a magneto electrode based on graphite-epoxy composite. The electrochemical detection is finally achieved by an enzyme marker, anti-digoxigenin horseradish peroxidase (HRP). The novel strategy was used for the rapid and sensitive detection of polymerase chain reaction (PCR) amplified samples. Promising resultants were also achieved for the DNA amplification directly performed on magnetic beads by using a novel magnetic primer, i.e., the up PCR primer bound to magnetic beads. Moreover, the magneto DNA biosensing assay was able to detect changes at single nucleotide polymorphism (SNP) level, when stringent hybridization conditions were used. The reliability of the assay was tested for Salmonella spp., the most important pathogen affecting food safety.