Utilization of the three high-throughput SNP genotyping methods, the GOOD assay, Amplifluor and TaqMan, in diploid and polyploid plants

The application of high-throughput SNP genotyping is a great challenge for many research projects in the plant genetics domain. The GOOD assay for mass spectrometry, Amplifluor and TaqMan are three methods that rely on different principles for allele discrimination and detection, specifically, primer extension, allele-specific PCR and hybridization, respectively. First, with the goal of assessing allele frequencies by means of SNP genotyping, we compared these methods on a set of three SNPs present in the herbicide resistance genes CSR, AXR1 and IXR1 of Arabidopsis thaliana. In this comparison, we obtained the best results with TaqMan based on PCR specificity, flexibility in primer design and success rate. We also used mass spectrometry for genotyping polyploid species. Finally, a combination of the three methods was used for medium- to high-throughput genotyping in a number of different plant species. Here, we show that all three genotyping technologies are successful in discriminating alleles in various plant species and discuss the factors that must be considered in assessing which method to use for a given application.     

Development of Multiplex Genotyping Method of Polymorphic Markers of Genes Associated with Cognitive Abilities

A developed method of multiplex genotyping of polymorphic markers of genes associated with cognitive abilities and neuropsychiatric diseases is based on multilocus PCR and MALDI-TOF mass spectrometry of DNA molecules. The frequencies of 32 single-nucleotide markers localized in 24 genes are analyzed in a sample of elderly people from the Russian population of Tomsk. The data obtained are compared with data for populations from the 1000 Genomes Project.     

Hierarchical high-throughput SNP genotyping of the human Y chromosome using MALDI-TOF mass spectrometry

We have established the use of a primer extension/mass spectrometry method (the PinPoint assay) for high-throughput SNP genotyping of the human Y chromosome. 118 markers were used to define 116 haplogroups and typing was organised in a hierarchical fashion. Twenty multiplex PCR/primer extension reactions were set up and each sample could be assigned to a haplogroup with only two to five of these multiplex analyses. A single aliquot of one enzyme was found to be sufficient for both PCR and primer extension. We observed 100% accuracy in blind validation tests. The technique thus provides a reliable, cost-effective and automated method for Y genotyping, and the advantages of using a hierarchical strategy can be applied to any DNA segment lacking recombination.     

Analysis of TPOX short tandem repeat locus with matrix-associated laser desorption/ionization time-of-flight-based restriction fragment mass polymorphism assay

Short tandem repeat (STR) loci are routinely analyzed by capillary electrophoresis. However, this method has several disadvantages, including long operational time, low throughput, and inaccuracy. As a result of the introduction of matrix-associated laser desorption/ionization time-of-flight (MALDI–TOF) and electrospray ionization (ESI), mass spectrometry has become an alternative method for genotyping polymorphic STR loci. Here we established a restriction fragment mass polymorphism (RFMP) assay for genotyping STR locus, TPOX, by typeIIS restriction endonuclease cleavage of polymerase chain reaction (PCR) amplicon followed by MALDI–TOF mass spectrometry. The resulting TPOX genotypes from this assay were in good agreement with the results from direct DNA sequencing and GeneScan assays. Our results showed that the RFMP assay is an accurate and high-throughput method for analyzing long DNA fragments such as STR markers. Further research with multiple STR loci may allow this assay to be used for diverse applications such as forensics, paternity tests, and detection of genetic disorders.     

Large-scale genotyping by mass spectrometry: experience, advances and obstacles

Single nucleotide polymorphism (SNP) genotyping has become a key technology for genetic studies. In recent years, matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry has emerged as a very powerful method for SNP genotyping. Here, we discuss our experience in implementing a high-throughput SNP genotyping facility based on MALDI, and the issues encountered in adapting this to large-scale genetic studies. Most of these issues are not specific to using MALDI approaches, and they will also serve as valuable pointers for establishing high-throughput genotyping with other methods.     

SNPkit: an efficient approach to systematic evaluation of candidate single nucleotide polymorphisms in public databases

There is widespread interest in devising genotyping methods for SNPs that are robust, inexpensive, and simple to perform. Although several high-throughput SNP genotyping technologies have been developed, including the oligonucleotide ligation assay, real-time PCR, and mass spectrometry, the issues of simplicity and cost-effectiveness have not been adequately addressed. Here we describe the application of a novel computer software package, SNPkit, which designs SNP genotyping assays based on a classical approach for discriminating alleles, restriction enzyme digestion. SNPkit can be used in genotyping assays for almost any SNPs including those that do not alter "natural" restriction sites. Using this method, 164 SNPs have been evaluated in DNA samples from 48 immortalized cell lines of randomly selected Chinese subjects. Sixty-two (37.8%) of the SNPs appeared to be common (frequencies of the minor alleles are > or = 5%) and were subsequently applied to a larger population-based sample. Overall, by using SNPkit, we have been able to validate and genotype accurately a large fraction of publicly available SNPs without sophisticated instrumentation.     

Single-nucleotide polymorphisms: analysis by mass spectrometry

Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry has evolved as a powerful method for analyzing nucleic acids. Here we provide protocols for genotyping single-nucleotide polymorphisms (SNPs) by MALDI based on PCR and primer extension to generate allele-specific products. Furthermore, we present three different approaches for sample preparation of primer-extension products before MALDI analysis and discuss their potential areas of application. The first approach, the 'GOOD' assay, is a purification-free procedure that uses DNA-modification chemistry, including alkylation of phosphorothioate linkages in the extension primers. The other two approaches use either solid-phase extraction or microarray purification for the purification of primer-extension products. Depending on the reaction steps of the various approaches, the protocols take about 6-8 hours.     

Genotyping single-nucleotide polymorphisms by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry

In recent years matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) has emerged as a very powerful method for genotyping single nucleotide polymorphisms. The accuracy, speed of data accumulation, and data structure are the major features of MALDI. Several SNP genotyping methods have been implemented with a high degree of automation and are being applied for large-scale association studies. Most methods for SNP genotyping using MALDI mass spectrometric detection and their potential application for high-throughput are reviewed here.     

A novel automated assay with dual-color hybridization for single-nucleotide polymorphisms genotyping on gold magnetic nanoparticle array

A high-throughput and cost-effective single-nucleotide polymorphism (SNP) genotyping method based on a gold magnetic nanoparticle (GMNP) array with dual-color hybridization has been designed. Biotinylated single-strand polymerase chain reaction (PCR) products containing the SNP locus were captured by the GMNPs that were coated with streptavidin. The GMNP array was fabricated by immobilizing single-stranded DNA (ssDNA)-GMNP complexes onto a glass slide using a magnetic field, and SNPs were identified with dual-color fluorescence hybridization. Three different SNP loci from 24 samples were genotyped successfully using this platform. This procedure allows the user to directly analyze the bead fluorescence to determine the SNP genotype, and it eliminates the need for background subtraction for signal determination. This method also bypasses tedious PCR purification and concentration procedures, and it facilitates large-scale SNP studies by using a method that is highly sensitive, simple, labor-saving, and potentially automatable.     

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.     

Microsatellite genotyping by primer extension and MALDI-ToF mass spectrometry

A novel method for genotyping microsatellite alleles using primer extensions and mass spectrometry analysis has been developed. Following PCR amplification of the target region, a genotyping primer, with its 3′ end directly flanking the microsatellite repeats, was extended by a mixture of dNTPs complementary to the nucleotides composing the microsatellite. The length and molecular weight of extended primers vary with the number of repeats present in the allele(s) under examination. The weights of extension products were determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) and used to identify genotypes on the basis of differential primer extension. This is a platform that is not gel based and is amenable to multiplexing and automation. The technique enables identification of heterozygous progeny in which alleles differ by a single trinucleotide repeat. The method is illustrated by genotyping a polymorphic microsatellite identified in an intron of the barleyMlo gene.     

Quantitative approach to single-nucleotide polymorphism analysis using MALDI-TOF mass spectrometry

Pooling of DNA samples before genotyping is a valuable means of streamlining large-scale genotyping efforts in disease association studies, single-nucleotide polymorphism (SNP) validation or mutant allele screening programs. In this report, we explore the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to quantitative analysis of SNPs. The measurements are based on MALDI-TOF MS analysis of primer extension assays performed on standard mixtures of pooled PCR products at several test loci. The inherent high molecular weight resolution of MALDI-TOF MS conveys high specificity and good signal-to-noise ratio for performing accurate quantitation. The methods described maximize the sensitivity and quantitative capacity of MALDI-TOF MS while preserving the throughput and economic advantages of the MALDI-TOF platform. Using the format described, we demonstrate that allele frequencies as low as 5% can be detected quantitatively and unambiguously.     

TaqMan-MGB 探针检测GSTP1外显子5SNP可行性研究

目的：评估TaqMan-MGB探针基因分型方法检测已知SNP的可行性,并与传统的PCR-RFLP方法比较。方法：高通量的TaqMan-MGB探针基因分型方法已被用来检测单核苷酸多态性（SNP）。在321倒样本中,同时用TaqMan-MGB探针基因分型方法和PCR—RFLP方法检测GSTP1外显子5SNP。结果：2种方法所得结果完全一致。野生型（AA）226例（70．4％）,杂合子（AG）92例（28．7％）,纯合突变型3例（O．9％）。结论：TaqMan-MGB探针基因分型方法是一种能快速、高度特异性、高度自动化检测SNP的方法。可用于大规模的基因分型。     

Multiplex SNP genotyping by MALDI-TOF mass spectrometry: Frequencies of 56 immune response gene SNPs in human populations

Single-nucleotide polymorphisms (SNPs) are the most common type of genetic polymorphisms. Despite the progress in sequencing and postgenomic technologies, targeted SNP genotyping continues to be in highest demand in the approach to human and medical genetics. In this work, we describe the application of multiple SNP genotyping by MALDI-TOF mass spectrometry for analysis of genetic diversity of immune response genes in human populations. It was shown that MALDI-TOF mass spectrometry is a rapid, accurate, and efficient method of medium-scale SNP genotyping. Allele frequencies of 56 SNPs in 41 genes implicated in the regulation of immune response were similar in four populations studied (Russians, Komi, Khanty, and Buryats). These populations had similar levels of genetic diversity and were clustered according to their geographic location. The cost efficiency of MALDI-TOF mass spectrometry was evaluated compared to real-time PCR technology.     

Single nucleotide polymorphism genotyping by on-line liquid chromatography-mass spectrometry in forensic science of the Y-chromosomal locus M9

A method is described for genotyping alleles of the Y-chromosomal locus M9, incorporating DNA extraction, amplification by polymerase chain reaction (PCR), sample purification by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and allele identification by on-line hyphenation to electrospray ionization mass spectrometry (ESI-MS). The alleles G and C were differentiated in 114 base pair amplicons on the basis of intact molecular mass measurements with a mass accuracy between 0.007 and 0.017%. The accuracy of mass determination was significantly reduced to less than 0.0036% upon amplification of a short, 61 bp fragment. The application of steep gradients of acetonitrile in 25 mM butyldimethylammonium bicarbonate not only enabled the efficient separation of non-target components from the PCR product in a monolithic, poly-(styrene-divinylbenzene)-based capillary column, but also allowed the high-throughput analysis of the PCR products with cycle times of 2 min. The new method was compared to a conventional restriction fragment length polymorphism assay with capillary gel electrophoretic analysis. In a blind study, 90 samples of unrelated individuals were genotyped. The high accuracy (<0.004%) and small relative standard deviation (<0.007%, n=20) of mass measurements, which enables even the differentiation of A and T alleles with a mass difference of 9 mass units, make IP-RP-HPLC-ESI-MS a potent tool for the routine characterization of SNPs in forensic science.     

High-throughput polymorphism screening and genotyping with high-density oligonucleotide arrays

A highly reliable and efficient technology has been developed for high-throughput DNA polymorphism screening and large-scale genotyping. Photolithographic synthesis has been used to generate miniaturized, high-density oligonucleotide arrays. Dedicated instrumentation and software have been developed for array hybridization, fluorescent detection, and data acquisition and analysis. Specific oligonucleotide probe arrays have been designed to rapidly screen human STSs, known genes and full-length cDNAs. This has led to the identification of several thousand biallelic single-nucleotide polymorphisms (SNPs). Meanwhile, a rapid and robust method has been developed for genotyping these SNPs using oligonucleotide arrays. Each allele of an SNP marker is represented on the array by a set of perfect match and mismatch probes. Prototype genotyping chips have been produced to detect 400, 600 and 3000 of these SNPs. Based on the preliminary results, using oligonucleotide arrays to genotype several thousand polymorphic loci simultaneously appears feasible.     

Detection of specific DNA from crude extracts of rice seed grains using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

To rapidly detect specific genes, crude extracts prepared from rice seed grains were used as templates for PCR, the PCR products were digested with restriction enzymes or urasil-DNA glycosylase, and then matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) was used to detect amplified DNA. It was possible to amplify small DNA fragments (50–60 bp), but not large ones (>200 bp), using crude extracts as the PCR template. This method can be completed within 1 h, including extractions, and is well suited to automation for high-throughput analyses.