Facile method for automated genotyping of single nucleotide polymorphisms by mass spectrometry

In the future, analysis of single nucleotide polymorphisms (SNPs) should become a powerful tool for many genetic applications in areas such as association studies, pharmacogenetics and traceability in the agro-alimentary sector. A number of technologies have been developed for high-throughput genotyping of SNPs. Here we present the simplified GOOD assay for SNP genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI). The simplified GOOD assay is a single-tube, purification-free, three-step procedure consisting of PCR, primer extension and phosphodiesterase II digestion followed by mass spectrometric analysis. Due to the application of charge-tag technology, no sample purification is required prior to the otherwise very impurity-sensitive MALDI analysis. The use of methylphosphonate containing primers and ddNTPs or α-S-ddNTPs together with a novel DNA polymerase derived from Thermotoga maritima for primer extension allow the fluent preparation of negatively charge-tagged, allele-specific products. A key feature of this polymerase is its preference for ddNTPs and α-S-ddNTPs over dNTPs. The simplified GOOD assay was run with automatic liquid handling at the lowest manageable volumes, automatic data acquisition and interpretation. We applied this novel procedure to genotyping SNPs of candidate genes for hypertension and cardiovascular disease.     

Full flexibility genotyping of single nucleotide polymorphisms by the GOOD assay

Recently a facile method for genotyping single nucleotide polymorphisms (SNPs) using MALDI mass spectrometry, termed the GOOD assay, was developed. It does not require any purification and is performed with simple liquid handling, thermal incubation and cycling steps. Although this method is well suited to automation and high-throughput analysis of SNPs, it did not allow full flexibility due to lack of certain reagents. A complete set of β-cyanoethyl phosphoramidites is presented herein that give this SNP genotyping method full sequence and multiplex capabilities. Applications to SNP genotyping in the prion protein gene, the β-2-adrenergic receptor gene and the angiotensin converting enzyme gene using the GOOD assay are demonstrated. Because SNP genotyping technologies are generally very sensitive to varying DNA quality, the GOOD assay has been stabilised and optimised for low quality DNA. A template extraction method is introduced that allows genotyping from tissue that was taken while placing an ear tag on an animal. This dramatically facilitates the application of genotyping to animal agricultural applications, as it demonstrates that expensive and cumbersome DNA extraction procedures prior to genotyping can be avoided.     

A novel procedure for efficient genotyping of single nucleotide polymorphisms

Due to the surge in interest in using single nucleotide polymorphisms (SNPs) for genotyping a facile and affordable method for this is an absolute necessity. Here we introduce a procedure that combines an easily automatable single tube sample preparation with an efficient high throughput mass spectrometric analysis technique. Known point mutations or single nucleotide polymorphisms are easily analysed by this procedure. It starts with PCR amplification of a short stretch of genomic DNA, for example an exon of a gene containing a SNP. By shrimp alkaline phosphatase digest residual dNTPs are destroyed. Allele-specific products are generated using a special primer, a conditioned set of α-S-dNTPs and α-S-ddNTPs and a fresh DNA polymerase in a primer extension reaction. Unmodified DNA is removed by 5′-phosphodiesterase digestion and the modified products are alkylated to increase the detection sensitivity in the mass spectrometric analysis. All steps of the preparation are simple additions of solutions and incubations. The procedure operates at the lowest practical sample volumes and in contrast to other genotyping protocols with mass spectrometric detection requires no purification. This reduces the cost and makes it easy to implement. Here it is demonstrated in a version using positive ion detection on described mutations in exon 17 of the amyloid precursor protein gene and in a version using negative ion detection on three SNPs of the granulocyte-macrophage colony stimulating factor gene. Preparation and analysis of SNPs is shown separately and simultaneously, thus demonstrating the multiplexibility of this genotyping procedure. The preparation protocol for genotyping is adapted to the conditions used for the SNP discovery method by denaturing HPLC, thus demonstrating a facile link between protocols for SNP discovery and SNP genotyping. Results corresponded unanimously with the control sequencing. The procedure is useful for high throughput genotyping as it is required for gene identification and pharmacogenomics where large numbers of DNA samples have to be analysed. We have named this procedure the ‘GOOD Assay’ for SNP analysis.     

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.     

Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA

Whole genome amplification (WGA) procedures such as primer extension preamplification (PEP) or multiple displacement amplification (MDA) have the potential to provide an unlimited source of DNA for large-scale genetic studies. We have performed a quantitative evaluation of PEP and MDA for genotyping single nucleotide polymorphisms (SNPs) using multiplex, four-color fluorescent minisequencing in a microarray format. Forty-five SNPs were genotyped and the WGA methods were evaluated with respect to genotyping success, signal-to-noise ratios, power of genotype discrimination, yield and imbalanced amplification of alleles in the MDA product. Both PEP and MDA products provided genotyping results with a high concordance to genomic DNA. For PEP products the power of genotype discrimination was lower than for MDA due to a 2-fold lower signal-to-noise ratio. MDA products were indistinguishable from genomic DNA in all aspects studied. To obtain faithful representation of the SNP alleles at least 0.3 ng DNA should be used per MDA reaction. We conclude that the use of WGA, and MDA in particular, is a highly promising procedure for producing DNA in sufficient amounts even for genome wide SNP mapping studies.     

SNP genotyping using alkali cleavage of RNA/DNA chimeras and MALDI time-of-flight mass spectrometry

Single nucleotide polymorphisms (SNPs) are now widely used for many DNA analysis applications such as linkage disequilibrium mapping, pharmacogenomics and traceability. Many methods for SNP genotyping exist with diverse strategies for allele-distinction. Mass spectrometers are used most commonly in conjunction with primer extension procedures with allele-specific termination. Here we present a novel concept for allele-preparation for SNP genotyping. Primer extension is carried out with an extension primer positioned immediately upstream of the SNP that is to be genotyped, a complete set of four ribonucleotides and a ribonucleotide incorporating DNA polymerase. The allele-extension products are then treated with alkali, which results in the cleavage immediately after the first added ribonucleotide. In addition, to obtain fragments easily detectable by mass spectrometry, we have included a ribonucleotide in the primer usually at the fourth nucleotide from the 3′ terminus. The method was tested on four SNPs each with a different combination of nucleotides. The advantage over other mass spectrometry-based SNP genotyping assays is that this one only requires a PCR, a primer extension reaction with a universal extension mix and an inexpensive facile cleavage reaction, which makes it overall very cost effective and easy in handling.     

High-throughput genotyping of single nucleotide polymorphisms using new biplex invader technology

The feasibility of large-scale genome-wide association studies of complex human disorders depends on the availability of accurate and efficient genotyping methods for single nucleotide polymorphisms (SNPs). We describe a new platform of the invader assay, a biplex assay, where both alleles are interrogated in a single reaction tube. The assay was evaluated on over 50 different SNPs, with over 20 SNPs genotyped in study cohorts of over 1500 individuals. We assessed the usefulness of the new platform in high-throughput genotyping and compared its accuracy to genotyping results obtained by the traditional monoplex invader assay, TaqMan genotyping and sequencing data. We present representative data for two SNPs in different genes (CD36 and protein tyrosine phosphatase 1β) from a study cohort comprising over 1500 individuals with high or low-normal blood pressure. In this high-throughput application, the biplex invader assay is very accurate, with an error rate of <0.3% and a failure rate of 1.64%. The set-up of the assay is highly automated, facilitating the processing of large numbers of samples simultaneously. We present new analysis tools for the assignment of genotypes that further improve genotyping success. The biplex invader assay with its automated set-up and analysis offers a new efficient high-throughput genotyping platform that is suitable for association studies in large study cohorts.     

Accessing single nucleotide polymorphisms in genomic DNA by direct multiplex polymerase chain reaction amplification on oligonucleotide microarrays

This study introduces a DNA microarray-based genotyping system for accessing single nucleotide polymorphisms (SNPs) directly from a genomic DNA sample. The described one-step approach combines multiplex amplification and allele-specific solid-phase PCR into an on-chip reaction platform. The multiplex amplification of genomic DNA and the genotyping reaction are both performed directly on the microarray in a single reaction. Oligonucleotides that interrogate single nucleotide positions within multiple genomic regions of interest are covalently tethered to a glass chip, allowing quick analysis of reaction products by fluorescence scanning. Due to a fourfold SNP detection approach employing simultaneous probing of sense and antisense strand information, genotypes can be automatically assigned and validated using a simple computer algorithm. We used the described procedure for parallel genotyping of 10 different polymorphisms in a single reaction and successfully analyzed more than 100 human DNA samples. More than 99% of genotype data were in agreement with data obtained in control experiments with allele-specific oligonucleotide hybridization and capillary sequencing. Our results suggest that this approach might constitute a powerful tool for the analysis of genetic variation.     

Multiplex SNP genotyping in pooled DNA samples by a four-colour microarray system

We selected 125 candidate single nucleotide polymorphisms (SNPs) in genes belonging to the human type 1 interferon (IFN) gene family and the genes coding for proteins in the main type 1 IFN signalling pathway by screening databases and by in silico comparison of DNA sequences. Using quantitative analysis of pooled DNA samples by solid-phase mini-sequencing, we found that only 20% of the candidate SNPs were polymorphic in the Finnish and Swedish populations. To allow more effective validation of candidate SNPs, we developed a four-colour microarray-based mini-sequencing assay for multiplex, quantitative allele frequency determination in pooled DNA samples. We used cyclic mini-sequencing reactions with primers carrying 5′-tag sequences, followed by capture of the products on microarrays by hybridisation to complementary tag oligonucleotides. Standard curves prepared from mixtures of known amounts of SNP alleles demonstrate the applicability of the system to quantitative analysis, and showed that for about half of the tested SNPs the limit of detection for the minority allele was below 5%. The microarray-based genotyping system established here is universally applicable for genotyping and quantification of any SNP, and the validated system for SNPs in type 1 IFN-related genes should find many applications in genetic studies of this important immunoregulatory pathway.     

Primer-design for multiplexed genotyping

Single-nucleotide polymorphism (SNP) analysis is a powerful tool for mapping and diagnosing disease-related alleles. Mutation analysis by polymerase-mediated single-base primer extension (minisequencing) can be massively parallelized using DNA microchips or flow cytometry with microspheres as solid support. By adding a unique oligonucleotide tag to the 5′ end of the minisequencing primer and attaching the complementary antitag to the array or bead surface, the assay can be ‘demultiplexed’. Such high-throughput scoring of SNPs requires a high level of primer multiplexing in order to analyze multiple loci in one assay, thus enabling inexpensive and fast polymorphism scoring. We present a computer program to automate the design process for the assay. Oligonucleotide primers for the reaction are automatically selected by the software, a unique DNA tag/antitag system is generated, and the pairing of primers and DNA tags is automatically done in a way to avoid any crossreactivity. We report results on a 45-plex genotyping assay, indicating that minisequencing can be adapted to be a powerful tool for high-throughput, massively parallel genotyping. The software is available to academic users on request.     

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.     

MARA: a novel approach for highly multiplexed locus-specific SNP genotyping using high-density DNA oligonucleotide arrays

We have developed a locus-specific DNA target preparation method for highly multiplexed single nucleotide polymorphism (SNP) genotyping called MARA (Multiplexed Anchored Runoff Amplification). The approach uses a single primer per SNP in conjunction with restriction enzyme digested, adapter-ligated human genomic DNA. Each primer is composed of common sequence at the 5′ end followed by locus-specific sequence at the 3′ end. Following a primary reaction in which locus-specific products are generated, a secondary universal amplification is carried out using a generic primer pair corresponding to the oligonucleotide and genomic DNA adapter sequences. Allele discrimination is achieved by hybridization to high-density DNA oligonucleotide arrays. Initial multiplex reactions containing either 250 primers or 750 primers across nine DNA samples demonstrated an average sample call rate of ~95% for 250- and 750-plex MARA. We have also evaluated >1000- and 4000-primer plex MARA to genotype SNPs from human chromosome 21. We have identified a subset of SNPs corresponding to a primer conversion rate of ~75%, which show an average call rate over 95% and concordance >99% across seven DNA samples. Thus, MARA may potentially improve the throughput of SNP genotyping when coupled with allele discrimination on high-density arrays by allowing levels of multiplexing during target generation that far exceed the capacity of traditional multiplex PCR.     

Rapid genotyping by MALDI-monitored nuclease selection from probe libraries

Data on five single-nucleotide polymorphisms (SNPs) per gene are estimated to allow association of disease risks or pharmacogenetic parameters with individual genes. Efficient technologies for rapidly detecting SNPs will therefore facilitate the mining of genomic information. Known methods for SNP analysis include restriction-fragment-length polymorphism polymerase chain reaction (PCR), allele-specific oligomer hybridization, oligomer-specific ligation assays, minisequencing, direct sequencing, fluorescence-detected 5'-exonuclease assays, and hybridization with PNA probes. Detection by mass spectrometry (MS) offers speed and high resolution. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) can detect primer extension products, mass-tagged oligonucleotides, DNA created by restriction endonuclease cleavage, and genomic DNA. We have previously reported MALDI-TOF-monitored nuclease selections of modified oligonucleotides with increased affinity for targets. Here we use nuclease selections for genotyping by treating DNA to be analyzed with oligonucleotide probes representing known genotypes and digesting probes that are not complementary to the DNA. With phosphodiesterase I, the target-bound, complementary probe is largely refractory to nuclease attack and its peak persists in mass spectra (Fig. 1A). In optimized assays, both alleles of a heterozygote were genotyped with six nonamer DNA probes (> or = 125 fmol each) and asymmetrically amplified DNA from exon 10 of the cystic fibrosis transmembrane regulatory gene (CFTR).     

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.     

Genotyping single nucleotide polymorphisms directly from genomic DNA by invasive cleavage reaction on microspheres

Here we report proof-of-principle for a microsphere-based genotyping assay that detects single nucleotide polymorphisms (SNPs) directly from human genomic DNA samples. This assay is based on a structure-specific cleavage reaction that achieves single base discrimination with a 5′-nuclease which recognizes a tripartite substrate formed upon hybridization of target DNA with probe and upstream oligonucleotides. The assay is simple with two easy steps: a cleavage reaction, which generates fluorescent signal on microsphere surfaces, followed by flow cytometry analysis of the microspheres. Genomic DNA samples were genotyped for the SNP in the Apolipoprotein E gene at amino acid position 158. The assay successfully scored wild type, heterozygous and homozygous mutants. To our knowledge, this is the first report of a solid-support assay for detection of SNPs directly from genomic DNA without PCR amplification of the target.     

Genotyping of Single Nucleotide Polymorphisms in DNA Isolated from Serum Using Sequenom MassARRAY Technology

Background

Large epidemiologic studies have the potential to make valuable contributions to the assessment of gene-environment interactions because they prospectively collected detailed exposure data. Some of these studies, however, have only serum or plasma samples as a low quantity source of DNA.

Methods

We examined whether DNA isolated from serum can be used to reliably and accurately genotype single nucleotide polymorphisms (SNPs) using Sequenom multiplex SNP genotyping technology. We genotyped 81 SNPs using samples from 158 participants in the NYU Women’s Health Study. Each participant had DNA from serum and at least one paired DNA sample isolated from a high quality source of DNA, i.e. clots and/or cell precipitates, for comparison.

Results

We observed that 60 of the 81 SNPs (74%) had high call frequencies (≥95%) using DNA from serum, only slightly lower than the 85% of SNPs with high call frequencies in DNA from clots or cell precipitates. Of the 57 SNPs with high call frequencies for serum, clot, and cell precipitate DNA, 54 (95%) had highly concordant (>98%) genotype calls across all three sample types. High purity was not a critical factor to successful genotyping.

Conclusions

Our results suggest that this multiplex SNP genotyping method can be used reliably on DNA from serum in large-scale epidemiologic studies.     

Large-scale genotyping of complex DNA

Genetic studies aimed at understanding the molecular basis of complex human phenotypes require the genotyping of many thousands of single-nucleotide polymorphisms (SNPs) across large numbers of individuals. Public efforts have so far identified over two million common human SNPs; however, the scoring of these SNPs is labor-intensive and requires a substantial amount of automation. Here we describe a simple but effective approach, termed whole-genome sampling analysis (WGSA), for genotyping thousands of SNPs simultaneously in a complex DNA sample without locus-specific primers or automation. Our method amplifies highly reproducible fractions of the genome across multiple DNA samples and calls genotypes at >99% accuracy. We rapidly genotyped 14,548 SNPs in three different human populations and identified a subset of them with significant allele frequency differences between groups. We also determined the ancestral allele for 8,386 SNPs by genotyping chimpanzee and gorilla DNA. WGSA is highly scaleable and enables the creation of ultrahigh density SNP maps for use in genetic studies.     

Multiplexed SNP genotyping using the Qbead™ system: a quantum dot-encoded microsphere-based assay

We have developed a new method using the Qbead™ system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot™ semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral ‘barcodes’ are created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein–protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications.     

SNP genotyping through the melting analysis of unlabelled oligonucleotide applied on dilute PCR amplicon

Adaptation of DNA melting analysis for polymorphic single nucleotides (SNPs) genotyping using an unlabeled oligonucleotide probe for polymorphic DNAs under the presence of fluorescent DNA binding dye necessitates a reaction condition where the probe efficiently associates with a target strand that is PCR amplified. We present experimental evidence that application of an unlabeled probe to a dilute PCR amplicon provides a condition such that the fluorescent signals gained subsequently by probe melting are sufficient to discriminate allelic identities. This approach is best exploited by adapting the multiplexing PCR technique in order to cover multiple SNPs for given samples. 3′-end modification of the probe is unnecessary as the amplicon dilution step provides a way of inactivating the polymerase through divalent cation chelation. With the use of low-cost reagents and ordinary laboratory equipment, this method offers a rapid, simple and cost-efficient way of SNP genotyping.     

Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry

BACKGROUND: We have developed a rapid, high throughput method for single nucleotide polymorphism (SNP) genotyping that employs an oligonucleotide ligation assay (OLA) and flow cytometric analysis of fluorescent microspheres. METHODS: A fluoresceinated oligonucleotide reporter sequence is added to a "capture" probe by OLA. Capture probes are designed to hybridize both to genomic "targets" amplified by polymerase chain reaction and to a separate complementary DNA sequence that has been coupled to a microsphere. These sequences on the capture probes are called "ZipCodes". The OLA-modified capture probes are hybridized to ZipCode complement-coupled microspheres. The use of microspheres with different ratios of red and orange fluorescence makes a multiplexed format possible where many SNPs may be analyzed in a single tube. Flow cytometric analysis of the microspheres simultaneously identifies both the microsphere type and the fluorescent green signal associated with the SNP genotype. RESULTS: Application of this methodology is demonstrated by the multiplexed genotyping of seven CEPH DNA samples for nine SNP markers located near the ApoE locus on chromosome 19. The microsphere-based SNP analysis agreed with genotyping by sequencing in all cases. CONCLUSIONS: Multiplexed SNP genotyping by OLA with flow cytometric analysis of fluorescent microspheres is an accurate and rapid method for the analysis of SNPs.