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.     

Quantitative heteroduplex analysis for single nucleotide polymorphism genotyping

High-resolution melting of polymerase chain reaction (PCR) products can detect heterozygous mutations and most homozygous mutations without electrophoretic or chromatographic separations. However, some homozygous single nucleotide polymorphism (SNPs) have melting curves identical to that of the wild-type, as predicted by nearest neighbor thermodynamic models. In these cases, if DNA of a known reference genotype is added to each unknown before PCR, quantitative heteroduplex analysis can differentiate heterozygous, homozygous, and wild-type genotypes if the fraction of reference DNA is chosen carefully. Theoretical calculations suggest that melting curve separation is proportional to heteroduplex content difference and that the addition of reference homozygous DNA at one seventh of total DNA results in the best discrimination between the three genotypes of biallelic SNPs. This theory was verified experimentally by quantitative analysis of both high-resolution melting and temperature-gradient capillary electrophoresis data. Reference genotype proportions other than one seventh of total DNA were suboptimal and failed to distinguish some genotypes. Optimal mixing before PCR followed by high-resolution melting analysis permits genotyping of all SNPs with a single closed-tube analysis.     

High-throughput single nucleotide polymorphism genotyping for breeding applications in rice using the BeadXpress platform

Multiplexed single nucleotide polymorphism (SNP) markers have the potential to increase the speed and cost-effectiveness of genotyping, provided that an optimal SNP density is used for each application. To test the efficiency of multiplexed SNP genotyping for diversity, mapping and breeding applications in rice (Oryza sativa L.), we designed seven GoldenGate VeraCode oligo pool assay (OPA) sets for the Illumina BeadXpress Reader. Validated markers from existing 1536 Illumina SNPs and 44?K Affymetrix SNP chips developed at Cornell University were used to select subsets of informative SNPs for different germplasm groups with even distribution across the genome. A 96-plex OPA was developed for quality control purposes and for assigning a sample into one of the five O. sativa population subgroups. Six 384-plex OPAs were designed for genetic diversity analysis, DNA fingerprinting, and to have evenly-spaced polymorphic markers for quantitative trait locus (QTL) mapping and background selection for crosses between different germplasm pools in rice: Indica/Indica, Indica/Japonica, Japonica/Japonica, Indica/O. rufipogon, and Japonica/O. rufipogon. After testing on a diverse set of rice varieties, two of the SNP sets were re-designed by replacing poor-performing SNPs. Pilot studies were successfully performed for diversity analysis, QTL mapping, marker-assisted backcrossing, and developing specialized genetic stocks, demonstrating that 384-plex SNP genotyping on the BeadXpress platform is a robust and efficient method for marker genotyping in rice.     

Multiplex single nucleotide polymorphism genotyping by adapter ligation-mediated allele-specific amplification

An improved approach for increasing the multiplex level of single nucleotide polymorphism (SNP) typing by adapter ligation-mediated allele-specific amplification (ALM-ASA) has been developed. Based on an adapter ligation, each reaction requires n allele-specific primers plus an adapter-specific primer that is common for all SNPs. Thus, only n+1 primers are used for an n-plex PCR amplification. The specificity of ALM-ASA was increased by a special design of the adapter structure and PCR suppression. Given that the genetic polymorphisms in the liver enzyme cytochrome P450 CYP2D6 (debrisoquine 4-hydroxylase) have profound effects on responses of individuals to a particular drug, we selected 17 SNPs in the CYP2D6 gene as an example for the multiplex SNP typing. Without extensive optimization, we successfully typed 17-plex SNPs in the CYP2D6 gene by ALM-ASA. The results for genotyping 70 different genome samples by the 17-plex ALM-ASA were completely consistent with those obtained by both Sanger's sequencing and PCR restriction fragment length polymorphism (PCR-RFLP) analysis. ALM-ASA is a potential method for SNP typing at an ultra-low cost because of a high multiplex level and a simple optimization step for PCR. High-throughput SNP typing could be readily realized by coupling ALM-ASA with a well-developed automation device for sample processing.     

Rapid characterization of DNA oligomers and genotyping of single nucleotide polymorphism using nucleotide-specific mass tags

Using currently available MS-based methods, accurate mass measurements are essential for the characterization of DNA oligomers. However, there is a lack of specificity in mass peaks when the characterization of individual DNA species in a mass spectrum is dependent solely upon the mass-to-charge ratio (m/z). Here, we utilize nucleotide-specific tagging with stable isotopes to provide internal signatures that quantitatively display the nucleotide content of oligomer peaks in MS spectra. The characteristic mass-split patterns induced by the partially ¹³C/¹⁵N-enriched dNTPs in DNA oligomers indicate the number of labeled precursors and in turn the base substitution in each mass peak, and provide for efficient SNP detection. Signals in mass spectra not only reflect the masses of particular DNA oligomers, but also their specific composition of particular nucleotides. The measurements of mass tags are relative in the mass-split pattern and, hence, the accuracy of the determination of nucleotide substitution is indirectly increased. For high sample throughput, ¹³C/¹⁵N-labeled sequences of interest have been generated, excised in solution and purified for MS analysis in a single-tube format. This method can substantially improve the specificity, accuracy and efficiency of mass spectrometry in the characterization of DNA oligomers and genetic variations.     

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.     

Multiplex single nucleotide polymorphism (SNP)-based genotyping in allohexaploid wheat using padlock probes

Single nucleotide polymorphisms are the most common polymorphism in plant and animal genomes and, as such, are the logical choice for marker-assisted selection. However, many plants are also polyploid, and marker-assisted selection can be complicated by the presence of highly similar, but non-allelic, homoeologous sequences. Despite this, there is practical and academic demand for high-throughput genotyping in several polyploid crop species, such as allohexaploid wheat. In this paper, we present such a system, which utilizes public single nucleotide polymorphisms previously identified in both agronomically important genes and in randomly selected, mapped, expressed sequence tags developed by the wheat community. To achieve relatively high levels of multiplexing, we used non-amplified genomic DNA and padlock probe pairs, together with high annealing temperatures, to differentiate between similar sequences in the wheat genome. Our results suggest that padlock probes are capable of discriminating between homoeologous sequences and hence can be used to efficiently genotype wheat varieties.     

A graphene-based platform for single nucleotide polymorphism (SNP) genotyping

A facile, rapid, stable and sensitive approach for fluorescent detection of single nucleotide polymorphism (SNP) is designed based on DNA ligase reaction and π-stacking between the graphene and the nucleotide bases. In the presence of perfectly matched DNA, DNA ligase can catalyze the linkage of fluorescein amidite-labeled single-stranded DNA (ssDNA) and a phosphorylated ssDNA, and thus the formation of a stable duplex in high yield. However, the catalytic reaction cannot effectively carry out with one-base mismatched DNA target. In this case, we add graphene to the system in order to produce different quenching signals due to its different adsorption affinity for ssDNA and double-stranded DNA. Taking advantage of the unique surface property of graphene and the high discriminability of DNA ligase, the proposed protocol exhibits good performance in SNP genotyping. The results indicate that it is possible to accurately determine SNP with frequency as low as 2.6% within 40 min. Furthermore, the presented flexible strategy facilitates the development of other biosensing applications in the future.     

Multiplex SNP-SCALE: a cost-effective medium-throughput single nucleotide polymorphism genotyping method

We describe a convenient, cost-effective and flexible medium-throughput single nucleotide polymorphism (SNP) genotyping method, Multiplex SNP-SCALE, which enables the simultaneous amplification by polymerase chain reaction (PCR) of up to 25 (or potentially more) loci followed by electrophoresis in an automated DNA sequencer. We extended the original SNP-SCALE method to include (i) use of a commercial multiplex PCR kit, (ii) a four-dye system, (iii) much-reduced (2-μL) reaction volumes, (iv) drying down of template DNA before PCR, (v) use of pig-tailed primers, (vi) a PCR product weighting system, (vii) a standard optimized touchdown PCR thermocycling programme, and (viii) software (SNP-SCALE Primer Designer) that automatically designs suitable SNP-SCALE primers for a batch of loci. This new protocol was validated for different types of SNPs. The method is cost- and time-effective for medium-scale evolutionary and ecological projects involving 10s to 100s of loci.     

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.     

High-throughput single nucleotide polymorphism genotyping in wheat (Triticum spp.)

Over the past few years, considerable progress has been made in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, largely through the investment of the human genetics community. These technologies are well adapted to diploid species. For plant breeding purposes, it is important to determine whether these genotyping methods are adapted to polyploidy, as most major crops are former or recent polyploids. To address this problem, we tested the capacity of the multiplex technology SNPlex™ with a set of 47 wheat SNPs to genotype DNAs of 1314 lines that were organized in four 384-well plates. These lines represented different taxa of tetra- and hexaploid Triticum species and their wild diploid relatives. We observed 40 markers which gave less than 20% missing data. Different methods, based on either Sanger sequencing or the MassARRAY^® genotyping technology, were then used to validate the genotypes obtained by SNPlex™ for 11 markers. The concordance of the genotypes obtained by SNPlex™ with the results obtained by the different validation methods was 96%, except for one discarded marker. Furthermore, a mapping study on six markers showed the expected genetic positions previously described. To conclude, this study showed that high-throughput genotyping technologies developed for diploid species can be used successfully in polyploids, although there is a need for manual reading. For the first time in wheat species, a core of 39 SNPs is available that can serve as the basis for the development of a complete SNPlex™ set of 48 markers.     

Suspension arrays for high throughput, multiplexed single nucleotide polymorphism genotyping

BACKGROUND: Genetic diversity can help explain disease susceptibility and differential drug response. The most common type of variant is the single nucleotide polymorphism (SNP). We present a low-cost, high throughput assay for SNP genotyping. METHODS: The assay uses oligonucleotide probes covalently attached to fluorescently encoded microspheres. These probes are hybridized directly to fluorescently labeled polymerase chain reaction (PCR) products and the results are analyzed in a standard flow cytometer. RESULTS: The genotypes determined with our assay are in good agreement with those determined by TaqMan. The range of G/C content for oligonucleotide probes was 23.5-65% in the 17 bases surrounding the SNP. Further optimization of probe length and target concentration is shown to dramatically enhance the assay performance for certain SNPs. Using microspheres which have unique fluorescent signatures, we performed a 32-plex assay where we simultaneously determined the genotypes of eight different polymorphic genes. CONCLUSIONS: We demonstrate, for the first time, the feasibility of multiplexed genotyping with suspension arrays using direct hybridization analyses. Our approach enables probes to be removed from or added to an array, enhancing flexibility over conventional chips. The ability to multiplex both the PCR preparation and the hybridization should enhance the throughput, cost, and speed of the assay.     

Improvement of single nucleotide polymorphism genotyping by allele-specific PCR using primers modified with an ENA residue

When we placed an ENA residue into primers at the 3' end, or the n-1, n-2, or n-3 position, which included a single nucleotide polymorphism (SNP) site at the 3' end, only primers containing the ENA residue at the n-2 position were read by Taq DNA polymerase for amplification. The use of the ENA primers avoided the generation of undesired short products, which are thought to be derived from primer-dimers. A greater discrimination of the SNP site by these primers containing the ENA residue was observed compared with that of the corresponding unmodified DNA primers that are often used for allele-specific polymerase chain reaction (AS-PCR). This improvement is probably due to the difficulty of incorporating a nucleotide into the mismatched ENA primer by Taq DNA polymerase in the modified primer-template duplex. These results demonstrate that ENA primer-based AS-PCR would enable a rapid and reliable technique for SNP genotyping.     

Thirty‐two single nucleotide polymorphism markers for high‐throughput genotyping of sockeye salmon

We characterize 32 single nucleotide polymorphism genotyping assays for resolving genotypic variation in sockeye salmon Oncorhynchus nerka in the Pacific Rim. These assays are based on the 5′‐nuclease reaction and thus facilitate high‐throughput genotyping with minimal optimization time. Minor allele frequency differences (Δq) among collections were between 4.7% and 97.9%, resulting in per locus F_ST estimates of 0.02–0.71 with an average of 0.22.     

Bayesian estimation of genomic copy number with single nucleotide polymorphism genotyping arrays

Background

The identification of copy number aberration in the human genome is an important area in cancer research. We develop a model for determining genomic copy numbers using high-density single nucleotide polymorphism genotyping microarrays. The method is based on a Bayesian spatial normal mixture model with an unknown number of components corresponding to true copy numbers. A reversible jump Markov chain Monte Carlo algorithm is used to implement the model and perform posterior inference.

Results

The performance of the algorithm is examined on both simulated and real cancer data, and it is compared with the popular CNAG algorithm for copy number detection.

Conclusions

We demonstrate that our Bayesian mixture model performs at least as well as the hidden Markov model based CNAG algorithm and in certain cases does better. One of the added advantages of our method is the flexibility of modeling normal cell contamination in tumor samples.     

Multiple displacement amplification prior to single nucleotide polymorphism genotyping in epidemiologic studies

We assessed the whole genome amplification strategy, known as multiple displacement amplification (MDA), for use with the TaqMan genotyping platform for DNA samples derived from two case-control studies nested in the Nurses' Health Study and the Physicians' Health Study. Our objectives were to (1) quantify DNA yield from samples of varying starting concentrations and (2) assess whether MDA products give an accurate representation of the original genomic sequence. Multiple displacement amplification yielded a mean 23000-fold increase in DNA quantity and genotyping results demonstrate 99.95% accuracy across six SNPs from four genes for 352 samples included in this study. These results suggest that MDA will provide a sufficiently robust amplification of limiting samples of genomic DNA that can be used for SNP genotyping in large case-control studies of complex diseases.     

Integrated allele-specific polymerase chain reaction-capillary electrophoresis microdevice for single nucleotide polymorphism genotyping

An integrated allele-specific (AS) polymerase chain reaction (PCR) and capillary electrophoresis (CE) microdevice has been developed for multiplex single nucleotide polymorphism (SNP) genotyping on a portable instrumentation, which was applied for on-site identification of HANWOO (Korean indigenous beef cattle). Twelve sets of primers were designed for targeting beef cattle's eleven SNP loci for HANWOO verification and one primer set for a positive PCR control, and the success rate for identification of HANWOO was demonstrated statistically. The AS PCR and CE separation for multiplex SNP typing was carried out on a glass-based microchip consisting of four layers: a microchannel plate for microfluidic control, a Pt-electrode plate for a resistance temperature detector (RTD), a poly(dimethylsiloxane) (PDMS) membrane and a manifold glass for microvalve function. The operation of the sample loading, AS PCR, microvalve, and CE on a chip was automated with a portable genetic analyzer, and the laser-induced fluorescence detection was performed on a miniaturized fluorescence detector. The blind samples were correctly identified as a HANWOO by showing one or two amplicon peaks in the electropherogram, while the imported beef cattle revealed more than five peaks. Our genetic analysis platform provides rapid, accurate, and on-site multiplex SNP typing.     

Recent patents on high-throughput single nucleotide polymorphism (SNP) genotyping methods

Single nucleotide polymorphisms (SNPs) are single-base inheritable variations in a given and defined genetic location that occur in at least 1% of the population. SNPs are useful markers for genetic association studies in disease susceptibility or adverse drug reactions, in evolutionary studies and forensic science. Given the potential impact of SNPs, the biotechnology industry has focused on the development of high-throughput methods for SNP genotyping. Many highthroughput SNP genotyping technologies are currently available and many others are being patented recently. Each offers a unique combination of scale, accuracy, throughput and cost. In this review, we described some of the most important recent SNP genotyping methods and also recent patents associated with it.     

Genotyping of single nucleotide polymorphism using model-based clustering

MOTIVATION: Single nucleotide polymorphisms have been investigated as biological markers and the representative high-throughput genotyping method is a combination of the Invader assay and a statistical clustering method. A typical statistical clustering method is the k-means method, but it often fails because of the lack of flexibility. An alternative fast and reliable method is therefore desirable. RESULTS: This paper proposes a model-based clustering method using a normal mixture model and a well-conceived penalized likelihood. The proposed method can judge unclear genotypings to be re-examined and also work well even when the number of clusters is unknown. Some results are illustrated and then satisfactory genotypings are shown. Even when the conventional maximum likelihood method and the typical k-means clustering method failed, the proposed method succeeded.