Genotyping of single-nucleotide polymorphisms by primer extension reaction in a dry-reagent dipstick format

The primer extension (PEXT) reaction is the most widely used approach to genotyping of single-nucleotide polymorphisms (SNPs). Current methods for analysis of PEXT reaction products are based on electrophoresis, fluorescence resonance energy transfer, fluorescence polarization, pyrosequencing, mass spectrometry, microarrays, and spectrally encoded microspheres. We report the first dry-reagent dipstick method that enables rapid visual detection of PEXT products without instrumentation. The method is applied to the analysis of six SNPs in the mannose-binding lectin gene (MBL2). The genomic region that spans each SNP of interest is amplified by PCR. Two primer extension reactions are performed with allele-specific primers (for one or the other variant nucleotide), which contain an oligo(dA) segment at the 5'-end. Biotin-dUTP is incorporated in the extended strand. The product is applied to the strip followed by immersion in the appropriate buffer. As the DNA moves along the strip by capillary action, it hybridizes with oligo(dT)-functionalized gold nanoparticles, such that only extended products are captured by immobilized streptavidin at the test zone, generating a red line. A second red line is formed at the control zone of the strip by hybridization of the nanoparticles with immobilized oligo(dA). The dipstick test is complete within 10 min. We analyzed six SNPs of the mannose-binding lectin gene (MBL2) using genomic DNA from 27 patients, representing a total of 74 variant nucleotide positions. Patient genotypes showed 100% concordance with direct DNA sequencing data. The described PEXT-dipstick assay is rapid and highly accurate; it does not require specialized instrumentation or highly trained technical personnel. It is appropriate for a diagnostic laboratory where a few selected SNP markers are examined per patient with a low cost per assay.     

A single base extension technique for the analysis of known mutations utilizing capillary gel electrophoreisis with electrochemical detection

A novel single nucleotide polymorphism (SNP) detection system is described in which the accuracy of DNA polymerase and advantages of electrochemical detection are demonstrated. A model SNP system is presented to illustrate the potential advantages in coupling the single base extension (SBE) technique to capillary gel electrophoresis (CGE) with electrochemical detection. An electrochemically labeled primer, with a ferrocene acetate covalently attached to its 5' end, is used in the extension reaction. When the Watson-Crick complementary ddNTP is added to the SBE reaction, the primer is extended by a single nucleotide. The reaction mixture is subsequently separated by CGE, and the ferrocene-tagged fragments are detected at the separation anode with sinusoidal voltammetry. This work demonstrates the first single base resolution separation of DNA coupled with electrochemical detection. The unextended primer (20-mer) and the 21-mer extension product are separated with a resolution of 0.8.     

Multiplexed,high-throughput genotyping by single-base extension and end-labeled free-solution electrophoresis

Technologies that allow for high-throughput, economical, and accurate single nucleotide polymorphism (SNP) genotyping are becoming crucial for modern genomic efforts. Here, we present a method for multiplexed single-base extension (SBE) genotyping that takes advantage of the unique separation modalities made possible via end-labeled free-solution electrophoresis (ELFSE). Three unique SBE oligonucleotide primers, which probe for mutations of clinical importance in the human p53 gene, were covalently conjugated to three unique polypeptoid frictional end labels and mixed together. This primer-polypeptoid conjugate cocktail was then used in a multiplexed SBE reaction followed by free-solution separation in a 96-capillary array electrophoresis (CAE) instrument. The study was designed to demonstrate multiplexed SNP genotyping of several loci in a single reaction and a single subsequent analysis. Further, the electrophoretic analysis was conducted without any viscous polymeric separation media, was complete in less than 10 min, and can be implemented in any capillary or microfluidic electrophoretic system with four-color fluorescent detection capabilities. Multiplexed SBE-ELFSE genotyping analysis resulted in the simultaneous and accurate genotyping of three p53 loci on five different DNA templates in a single reaction set and single CAE analysis. With the implementation of this method in 96 or more capillaries in parallel, high-throughput screening of SNPs will be accessible to a large number of laboratories.     

MALDI-TOF mass spectrometric detection of multiplex single base extended primers. A study of 17 y-chromosome single-nucleotide polymorphisms

One of the most promising techniques for typing of multiple single-nucleotide polymorphism (SNP) is detection of single base extension primers (SBE) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We present a new MALDI-TOF MS protocol for typing of multiple SNPs in a single reaction. Biotin-labeled ddNTPs were used in the SBE reaction and solid phase-bound monomeric avidin was used as capturing/purification scheme allowing the exclusive release of the SBE products under gentle conditions using 5% triethylamine. We dubbed this method monomeric avidin triethylamine purification. The biotin-labeled ddNTPs contained linkers with different masses ensuring a clear separation of the alleles even for SBE primers with a mass of 10 300 Da. Furthermore, only 25-350 fmol of SBE primers were necessary in order to obtain reproducible MALDI-TOF spectra. Similar signal intensities were obtained in the 5500-10 300 m/z mass range by increasing the concentration of the longer SBE primers in the reaction. To validate the technique, 17 Y-chromosome SNPs were analyzed in 200 males. The precision and accuracy of the mass determination were analyzed by parametric statistic, and the potential use of MALDI-TOF MS for SNP typing is discussed.     

High-throughput SNP genotyping by SBE/SBH

Despite much progress over the past decade, current single nucleotide polymorphism (SNP) genotyping technologies still offer an insufficient degree of multiplexing when required to handle user selected sets of SNPs. In this paper we propose a new genotyping assay architecture combining multiplexed solution-phase single-base extension (SBE) reactions with sequencing by hybridization (SBH) using universal DNA arrays such as all k-mer arrays. Simulation results on datasets both randomly generated and extracted from the NCBI dbSNP database suggest that the SBE/SBH architecture provides a flexible and cost-effective alternative to genotyping assays currently used in the industry, enabling genotyping of up to hundreds of thousands of SNPs per assay     

Single-nucleotide polymorphism genotyping by nanoparticle-enhanced surface plasmon resonance imaging measurements of surface ligation reactions

A sensitive method for the analysis of single nucleotide polymorphisms (SNPs) in genomic DNA that utilizes nanoparticle-enhanced surface plasmon resonance imaging (SPRI) measurements of surface enzymatic ligation reactions on DNA microarrays is demonstrated. SNP identification was achieved by using sequence-specific surface reactions of the enzyme Taq DNA ligase, and the presence of ligation products on the DNA microarray elements was detected using SPRI through the hybridization adsorption of complementary oligonucleotides attached to gold nanoparticles. The use of gold nanoparticles increases the sensitivity of the SPRI so that single bases in oligonucleotides can be successfully identified at a concentration of 1 pM. This sensitivity is amply sufficient for performing multiplexed SNP genotyping by using multiple PCR amplicons and should also allow for the direct detection and identification of SNP sequences from 1 pM unamplified genomic DNA samples with this array-based and label-free SPRI methodology. As a first example of SNP genotyping, three different human genomic DNA samples were screened for a possible point mutation in the BRCA1 gene that is associated with breast cancer.     

Homogeneous label-free genotyping of single nucleotide polymorphism using ligation-mediated strand displacement amplification with DNAzyme-based chemiluminescence detection

Genotyping of single nucleotide polymorphisms (SNPs) is a central challenge in disease diagnostics and personalized medicine. A novel label-free homogeneous SNP genotyping technique is developed on the basis of ligation-mediated strand displacement amplification (SDA) with DNAzyme-based chemiluminescence detection. Discrimination of single-base mismatches is first accomplished using DNA ligase to generate a ligation product between a discriminant probe and a common probe. The ligated product then initiates two consecutive SDA reactions to produce a great abundance of aptamer sequences against hemin, which can be probed by chemiluminscence detection. The developed strategy is demonstrated using a model SNP target of cytochrome P450 monooxygenase CYP2C19*2, a molecular marker for personalized medicines. The results reveal that the developed technique displays superb selectivity in discriminating single-base mismatches, very low detection limit as low as 0.1 fM, a wide dynamic range from 1 fM to 1 nM, and a high signal-to-background ratio of 150. Due to its label-free, homogeneous, and chemiluminescence-based detection format, this technique can be greatly robust, cost-efficient, readily automated, and scalable for parallel assays of hundreds of samples. The developed genotyping strategy might provide a robust, highly sensitive, and specific genotyping platform for genetic analysis and molecular diagnostics.     

Parallel single nucleotide polymorphism genotyping by surface invasive cleavage with universal detection

Large-scale investigations of sequence variation within the human species will provide information about the basis of heritable variation in disease susceptibility and human migration. The surface invader assay (an adaptation of the invasive cleavage reaction to an array format) is capable of exquisitely sensitive and specific detection of genetic variation. It is shown here that this genotyping technology can be multiplexed in a DNA array format, permitting the parallel analysis of a panel of single nucleotide polymorphisms (SNPs) directly from an unamplified genomic DNA target. In addition, a "universal" mode of detection was developed that makes use of a mixture of degenerate templates for DNA ligation to the surface-bound cleaved oligonucleotides and thereby makes this strategy amenable to any desired SNP site or combination of SNP sites, without regard to their particular DNA sequences. This approach was demonstrated on a proof-of-principle scale using small DNA arrays to genotype 6 SNP markers in the PTPN1 gene and 10 mutations in the cystic fibrosis transmembrane conductance regulator gene. This ability to analyze many different genetic variations in parallel, directly from unamplified human genomic DNA samples, lays the groundwork for the development of high-density arrays able to analyze hundreds of thousands or even millions of SNPs.     

Simple multiplex genotyping by surface-enhanced resonance Raman scattering

The accurate detection of DNA sequences is essential for a variety of post human genome projects including detection of specific gene variants for medical diagnostics and pharmacogenomics. A specific DNA sequence detection assay based on surface-enhanced resonance Raman scattering (SERRS) and an amplification refractory mutation system (ARMS) is reported. Initially, generation of PCR products was achieved by using specifically designed allele-specific SERRS active primers. Detection by SERRS of the PCR products confirmed the presence of the sequence tested for by the allele-specific oligonucleotides. This lead directly to the multiplex genotyping of human DNA samples for the deltaF508 mutational status of the cystic fibrosis transmembrane conductance regulator gene using SERRS active primers in an ARMS assay. Removal of the unincorporated primers allowed fast and accurate analysis of the three genotypes possible in this system in a multiplex format without any separation of amplicons. The results indicate that SERRS can be used in modern genetic analysis and offers an opportunity for the development of novel assays. This is the first demonstration of the use of SERRS in multiplex genotyping and shows potential advantages over fluorescence as a detection technique with considerable promise for future development.     

Single nucleotide polymorphism analysis based on minisequencing coupled with a fluorescence microsphere technology

In this paper, we describe a new method for detection of single nucleotide polymorphisms (SNPs) by applying the minisequencing principle to a fluorescence microsphere format. The specific primer, which was designed to anneal to its target of genomic DNA fragment immediately upstream of the polymorphic site, was immobilized to carboxylated Luminex microspheres as a probe. The primer was hybridized with genomic DNA fragments containing polymorphic sites and extended one base in the presence of biotin labeled ddNTP and DNA polymerase. After the extension reaction, Streptavidin-phycoerythrin was added to a reaction mixture to combine the biotin labeled with ddNTP. The final reaction products were analyzed by a Luminex 100 instrument. The fluorescence intensity of the Streptavidin-phycoerythrin combined with the extended ddNTP-biotin was used to identify the SNPs. The results showed that this method is highly sensitive, specific, and suitable for quantitative SNP detection. There was a good linear relationship between the mutant allele frequencies and the relative fluorescence intensities produced by mutant and wild-type gene fragments. A mutant allele frequency as low as 1.0% was accurately determined.     

Single nucleotide polymorphism genotyping and point mutation detection by ligation on microarrays

Based on the principle of sequencing by ligation, a novel method referred to as "Minisequencing by ligation" for detecting known single nucleotide variant in high-throughput assay formats is reported in this article. By designing a sequencing primer and fluorescently labeled probes each with a "discriminating" base according to the single base variation, this method can be used to analyze single nucleotide polymorphism (SNP) or point mutation in a number of samples in parallel. In our current study, three known nucleotides at a given position of three DNA templates with different CG contents are firstly genotyped on aldehyde-modified slide to testify the feasibility and optimize the reaction conditions. Then, by performing ligation reaction between phosphorylated 5' end of the sequencing primer and hydroxylated 3' end of the labeled probe or phosphorylated 5' end of the labeled probe and hydroxylated 3' end of the sequencing primer, a SNP locus (rs1800497 (C/T)) and a point mutation (Mt1555 (A/G)) have been accurately detected on aldehyde-modified microarray and Sepharose beads in acrylamide gel, respectively. It has demonstrated that "minisequencing by ligation," as a promising methodology, can perform point mutation and SNP analysis in a simple, cost-effective, robust and high-throughput way.     

Fabrication and application of single nucleotide polymorphisms library on magnetic nanoparticles using adaptor PCR

We have developed a novel approach to fabricate single nucleotide polymorphisms (SNPs) library on magnetic nanoparticles (MNPs) based on adaptor PCR. Each SNP locus in the library was interrogated by hybridization with a pair of allele specific dual-color fluorescence (Cy3, Cy5) probes to determine SNP. Two SNPs loci (M235T and A-6G) associated with essential hypertension in the angiotensinogen (AGT) gene were detected by this method and their fluorescent signals were quantified. The fluorescent ratios (match probe: mismatch probe signal) of homozygous genotypes were over 3.0, whereas heterozygous genotypes had ratios near to 1.0. Without any complex multiplex PCR procedure, it is a simple, efficient and reliable method for the multiplex SNPs detection using limited amount of DNA samples from individuals.     

Informative SNP selection methods based on SNP prediction

The search for the association between complex diseases and single nucleotide polymorphisms (SNPs) or haplotypes has recently received great attention. For these studies, it is essential to use a small subset of informative SNPs, i.e., tag SNPs, accurately representing the rest of the SNPs. Tag SNP selection can achieve: 1) considerable budget savings by genotyping only a limited number of SNPs and computationally inferring all other SNPs or 2) necessary reduction of the huge SNP sets (obtained, e.g., from Affymetrix) for further fine haplotype analysis. In this paper, we show that the tag SNP selection strongly depends on how the chosen tags will be used-advantage of one tag set over another can only be considered with respect to a certain prediction method. We show how to separate tag selection from SNP prediction and propose greedy and local-minimization algorithms for tag SNP selection. We give two novel approaches to SNP prediction based on multiple linear regression (MLR) and support vector machines (SVMs). An extensive experimental study on various datasets including ten regions from hapMap project shows that the MLR prediction combined with stepwise tag selection uses fewer tags than the state-of-the-art method of Halperin The MLR-based method also uses on average 30% fewer tags than IdSelect for statistical covering all SNPs. The tag selection based on SVM SNP prediction uses fewer tags to achieve the same prediction accuracy as the methods of Halldorsson     

A novel single nucleotide polymorphisms detection sensors based on magnetic nanoparticles array and dual-color single base extension

To fulfill the increasing need for large-scale genetic research, a high-throughput and automated SNPs genotyping method based on gold coated magnetic nanoparticles (GMNPs) array and dual-color single base extension has been designed. Biotinylated single base extension primer were captured onto the GMNPs coated with streptavidin. Dual-color fluorescence single base extension was employed as a tool for SNPs identification, and then a "bead array" was fabricated by spotting GMNPs with fluorophore on the glass slide. Using this platform, MTHFR gene C677T locus from 12 samples was genotyped. The results showed that all homozygous samples gave a signal/noise ratio over 30, and the fluorescent intensities ratios of heterozygote samples were around 1. With advantages to defeat the background subtract feature and read-out the fluorescence values of the GMNPs directly to determine their genotypes, without the necessary procedures for purification and complex reduction of PCR products, the application of this strategy to large-scale SNP studies will be simple, labor-saving, high sensitive and potential for automation.     

Terminal protection of small molecule-linked DNA: A versatile biosensor platform for protein binding and gene typing assay

Assays of small molecule-protein interactions are of tremendous importance in chemical genetics, molecular diagnostics, and drug development. This work reports a new finding of generalized terminal protection that small molecule-DNA chimeras are protected from degradation by various DNA exonucleases, when the small molecule moieties are bound to their protein targets. This generalization converts small molecule-protein interaction assays into the detection of DNA of various structures, affording a useful mechanism for the analytics of small molecules. On the basis of this mechanism, a label-free biosensor strategy has been developed for a homogeneous assay of protein-small molecule interactions based on the fluorescence staining detection. Also, a label-free SNP genotyping technique is proposed based on polymerase extension of a single nucleotide with a small molecule label. The developed techniques are demonstrated using a model protein-small molecule system of biotin/streptavidin and a model SNP system of human β-globin gene around the position of codon 39. The results revealed that the protein-small molecule interaction assay strategy shows dynamic responses in the concentration range from 0.5 to 100 nM with a detection limit of 0.1 nM, and the SNP typing technique gives dynamic responses in the concentration range from 0.1 to 200 nM with a detection limit of 0.02 nM. Besides desirable sensitivity, the developed strategies also offer high selectivity, excellent reproducibility, low cost, and simplified operations, implying that these techniques may hold considerable potential for molecular diagnostics and genomic research.     

DNA Nanotweezers and Graphene Transistor Enable Label‐Free Genotyping

Electronic DNA‐biosensor with a single nucleotide resolution capability is highly desirable for personalized medicine. However, existing DNA‐biosensors, especially single nucleotide polymorphism (SNP) detection systems, have poor sensitivity and specificity and lack real‐time wireless data transmission. DNA‐tweezers with graphene field effect transistor (FET) are used for SNP detection and data are transmitted wirelessly for analysis. Picomolar sensitivity of quantitative SNP detection is achieved by observing changes in Dirac point shift and resistance change. The use of DNA‐tweezers probe with high‐quality graphene FET significantly improves analytical characteristics of SNP detection by enhancing the sensitivity more than 1000‐fold in comparison to previous work. The electrical signal resulting from resistance changes triggered by DNA strand‐displacement and related changes in the DNA geometry is recorded and transmitted remotely to personal electronics. Practical implementation of this enabling technology will provide cheaper, faster, and portable point‐of‐care molecular health status monitoring and diagnostic devices.     

Quantification of relative gene dosage by single-base extension and high-performance liquid chromatography: application to the SMN1/SMN2 gene

One of the most commonly used techniques for genotyping of single-nucleotide polymorphism (SNP) is detection of single-base extensions (SBEs). We present a new, rapid, simple, and highly reliable method for accurate quantification of SNP variants in a single reaction. Our approach is based on SBE detection coupled with high-performance liquid chromatography (HPLC) quantification. To demonstrate the utility of our approach, we report data to determine the gene dosage for relative amounts of alleles in a homologous gene, allowing detection of mutation causing exon skipping in human SMN genes to determine the ratio between the copy numbers of the SMN1/SMN2 gene. We successfully determined the relative ratio of the SMN1 and SMN2 genes and showed assay characteristics using the SBE reaction coupled with HPLC. This assay approach readily scaled to high parallelization with multiplex SBE reactions in a single sample screened in one analysis. By screening for particular SNP genotypes, this assay can be used to determine the relative gene dosage that correlates highly with the patient's disease state. The next challenge is to apply this novel methodology in a clinical screening and quantification setting for special gene regions within highly homologous genes.     

Multiplex mass spectrometric genotyping of single nucleotide polymorphisms employing pyrrolidinyl peptide nucleic acid in combination with ion-exchange capture

A new ion-exchange capture technique is introduced for label-free sample preparation in single nucleotide polymorphism (SNP) genotyping. The DNA sample is hybridized with a new pyrrolidinyl peptide nucleic acid (PNA) probe and treated with a strong anion exchanger. The complementary PNA.DNA hybrid is selectively captured by the anion exchanger in the presence of noncomplementary or unhybridized PNA, allowing direct detection of the hybridization event on the anion exchanger by MALDI-TOF mass spectrometry after simple washing. The high specificity of the pyrrolidinyl PNA allows simultaneous multiplex SNP typing to be carried out at room temperature without the need for enzyme treatment or heating. Exemplary applications of this technique, in the identification of meat species in feedstuffs and in multiplex SNP typing of the human IL-10 gene promoter region are demonstrated, clearly suggesting the potential for much broader applications.     

Cationic comb-type copolymers for DNA analysis

Genetic diagnoses, such as single nucleotide polymorphism (SNP) typing, allow elucidation of gene-based physiological differences, such as susceptibility to diseases and response to drugs, among individuals. Many detection technologies, including allele-specific hybridization, allele-specific primer extension and oligonucleotide ligation, are being used to discriminate SNP alleles. These methods still have many unsolved practical issues. In general they require adequate and specific hybridizations of primer or probe DNAs with target DNAs. This frequently needs optimization of the probe/primer structures and operating conditions. In nature, highly homology-sensitive hybridization is assisted by a nucleic acid chaperone that reduces the energy barrier associated with breakage and reassociation of nucleic base pairs. Here we report a simple, quick, precise but enzyme-free method for SNP analysis. The method uses cationic comb-type copolymers (CCCs) producing high nucleic acid chaperone activities. A single-base mismatch in 20-mer DNA can be detected within a few minutes at ambient temperatures (25-37 degrees C). Even without careful optimization processes, the method has the sensitivity to detect the mismatches causing subtle changes (Delta T(m) equals approximately 1 degree C) in duplex thermal stability. CCCs may have various bioanalytical applications where precise hybridization of nucleic acids is needed.     

Multiplexed p53 mutation detection by free-solution conjugate microchannel electrophoresis with polyamide drag-tags

We report a new, bioconjugate approach to performing highly multiplexed single-base extension (SBE) assays, which we demonstrate by genotyping a large panel of point mutants in exons 5-9 of the p53 gene. A series of monodisperse polyamide "drag-tags" was created using both chemical and biological synthesis and used to achieve the high-resolution separation of genotyping reaction products by microchannel electrophoresis without a polymeric sieving matrix. A highly multiplexed SBE reaction was performed in which 16 unique drag-tagged primers simultaneously probe 16 p53 gene loci, with an abbreviated thermal cycling protocol of only 9 min. The drag-tagged SBE products were rapidly separated by free-solution conjugate electrophoresis (FSCE) in both capillaries and microfluidic chips with genotyping accuracy in excess of 96%. The separation requires less than 70 s in a glass microfluidic chip, or about 20 min in a commercial capillary array sequencing instrument. Compared to gel electrophoresis, FSCE offers greater freedom in the design of SBE primers by essentially decoupling the length of the primer and the electrophoretic mobility of the genotyping products. FSCE also presents new possibilities for the facile implementation of SBE on integrated microfluidic electrophoresis devices for rapid, high-throughput genetic mutation detection or SNP scoring.