Rapid single nucleotide polymorphism analysis by primer extension and capillary electrophoresis using polyvinyl pyrrolidone matrix

Rapid molecular diagnosis of 21-hydroxylase deficiency by detecting the most common mutation in the 21-hydroxylase gene is presented using primer extension and capillary electrophoresis with a polyvinyl pyrrolidone matrix. DNA samples were subjected to polymerase chain reaction (PCR) in order to amplify a 422 bp fragment of the CYP21 gene containing the single nucleotide polymorphism (SNP) site. This product served as a template in the primer extension reaction using a fluorescently labeled primer in close proximity to the SNP. ddGTP was used to block the extension if the mutation was present and the other three dNTPs to enable elongation of the primer. Fast analysis of the resulting fragments was performed by capillary electrophoresis using 10% polyvinylpyrrolidone as sieving and wall coating matrix. The Cy5-labeled primer and the two possible primer extension products (mutant and wild type) were completely separated in 90 s.     

Genotyping of single nucleotide polymorphisms by primer extension reaction and a dual-analyte bio/chemiluminometric assay

Primer extension reaction (PEXT) is the most widely used approach to genotyping of single nucleotide polymorphisms (SNP). It is based on the high accuracy of nucleotide incorporation by the DNA polymerase. We propose a dual-analyte bio/chemiluminometric method for the simultaneous detection of the PEXT reaction products of the normal and mutant allele in a high sample-throughput format. PCR-amplified DNA fragments that span the SNP of interest are subjected to two PEXT reactions using normal and mutant primers in the presence of digoxigenin-dUTP and biotin-dUTP. Both primers contain a d(A)₃₀ segment at the 5′-end but differ in the final nucleotide at the 3′-end. Under optimized conditions only the primer that is perfectly complementary with the interrogated DNA will be extended by DNA polymerase and lead to a digoxigenin- or biotin-labeled product. The products of the PEXT reactions are mixed, denatured, and captured in microtiter wells through hybridization with immobilized oligo(dT) strands. Detection is performed by adding a mixture of antidigoxigenin–alkaline phosphatase (ALP) conjugate and a streptavidin–aequorin conjugate. The flash-type bioluminescent reaction of aequorin is triggered by the addition of Ca²⁺. ALP is then measured by adding the appropriate chemiluminogenic substrate. The method was evaluated by genotyping two SNPs of the human mannose-binding lectin gene (MBL2) and one SNP of the cytochrome P450 gene CYP2D6. Patient genotypes showed 100% concordance with direct DNA sequencing data.     

Analysis of single nucleotide polymorphisms by primer extension and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method for typing single nucleotide polymorphisms (SNPs) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described, in which a mass-tagged dideoxynucleoside triphosphate is employed in a primer extension reaction in place of an unmodified dideoxynucleoside triphosphate (ddNTP). The increased mass difference due to the presence of the mass-tag greatly facilitates the accurate identification of the added nucleotide, and is particularly useful for typing heterozygous samples. Twenty commercially available mass-tagged dideoxynucleoside triphosphates were screened for amenability to incorporation by AmpliTaq FS and ThermoSequenase DNA polymerases in single nucleotide primer extension (SNuPE) reactions. Several sample preparation and purification methods were also examined and compared. Float dialysis was found to be a simple, versatile, and effective method for purification of the extension products. High specificity and sensitivity were obtained, and all six possible biallelic SNP heterozygotes were determined accurately using a 44-mer synthetic oligonucleotide target DNA as a model system. Further validation of the method was demonstrated in the analysis of five single-base mutations in exon IV of the human tyrosinase gene. Single nucleotide variations within 182-bp PCR amplicons amplified from three plasmid and three human genomic DNA samples were genotyped at five variable positions, with results in 100% concordance with conventional sequencing. Genotypes were determined accurately at five sequence-tagged sites (STSs).     

Short peptides enhance single cell adhesion and viability on microarrays

Single cell patterning holds important implications for biology, biochemistry, biotechnology, medicine, and bioinformatics. The challenge for single cell patterning is to produce small islands hosting only single cells and retaining their viability for a prolonged period of time. This study demonstrated a surface engineering approach that uses a covalently bound short peptide as a mediator to pattern cells with improved single cell adhesion and prolonged cellular viability on gold patterned SiO2 substrates. The underlying hypothesis is that cell adhesion is regulated by the type, availability, and stability of effective cell adhesion peptides, and thus covalently bound short peptides would promote cell spreading and, thus, single cell adhesion and viability. The effectiveness of this approach and the underlying mechanism for the increased probability of single cell adhesion and prolonged cell viability by short peptides were studied by comparing cellular behavior of human umbilical cord vein endothelial cells on three model surfaces whose gold electrodes were immobilized with fibronectin, physically adsorbed Arg-Glu-Asp-Val-Tyr, and covalently bound Lys-Arg-Glu-Asp-Val-Tyr, respectively. The surface chemistry and binding properties were characterized by reflectance Fourier transform infrared spectroscopy. Both short peptides were superior to fibronectin in producing adhesion of only single cells, whereas the covalently bound peptide also reduced apoptosis and necrosis of adhered cells. Controlling cell spreading by peptide binding domains to regulate apoptosis and viability represents a fundamental mechanism in cell-materials interaction and provides an effective strategy in engineering arrays of single cells.     

Genotyping using single nucleotide polymorphism, fluorescence spectroscopy and pattern recognition

This paper describes a method for genetic screening using single nucleotide polymorphism. Fluorescence spectra with an excitation frequency of 488 nm are recorded over a range of 550 to 660 nm of fragments of human DNA together with two fluorescent probe dyes attached to specific primers, one for each type of allele and a background dye, prepared using the Taqman reaction. The fluorescence spectra are monitored and principal components analysis used to separate spectra into three groups, which are visually identified as allele 1 (wild type), allele 2 (mutant) and mixed allele by comparison to reference samples. Malahanobis distance using 4 principal components are used to correctly classify samples into groups.     

Nanocrystal-based bioelectronic coding of single nucleotide polymorphisms

A bioelectronic method for coding unknown single nucleotide polymorphisms (SNPs) based on the use of different encoding nanocrystals is described. Four such nanocrystals, ZnS, CdS, PbS, and CuS, linked to the adenosine, cytidine, guanosine and thymidine mononucleotides, respectively, are sequentially introduced to the DNA hybrid-coated magnetic-bead solution. Each mutation captures via base pairing different nanocrystal-mononucleotide conjugates, and yields a characteristic multipotential voltammogram, whose peak potentials reflect the identity of the mismatch. The mismatch recognition events are being amplified by the metal accumulation feature of the stripping voltammetric transduction mode. Each of the eight possible one-base mismatches can thus be identified in a single voltammetric run. The use of nanocrystal tracers for detecting two known mutations in a single DNA target is also illustrated in connection to nanocrystals linked to two nucleotides along with a single voltammetric run. The protocol presented should facilitate the rapid, simple, low-cost, and high throughput screening for SNPs.     

A standard protocol for single nucleotide primer extension in the human genome using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Analysis of single nucleotide polymorphisms (SNPs) has become an increasingly important area of research, with numerous applications in medical genetics, population genetics, forensic science, and agricultural biotechnology. Large-scale SNP analyses require the development of methodologies that are economical, flexible, accurate and capable of automation. Primer extension in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is currently emerging as a potential method for high-throughput SNP genotyping. We have evaluated a number of published primer extension methods and refined a simple and robust protocol to analyze human autosomal disease-causing mutations and population genetic markers on the Y-chromosome. Twelve different variant sites were examined, and homozygotes, heterozygotes and hemizygotes were accurately typed. A 100% concordance was observed between SNP genotypes obtained using the MALDI-TOFMS technique and alternative genotyping methods, such as restriction fragment length polymorphism (RFLP) assays and denaturing high-performance liquid chromatography (DHPLC). Since multiple polymorphisms can be detected in single reactions, the method provides a cost-effective approach for SNP analysis. The protocol is also extremely flexible (able to accommodate new markers) and can be adapted to a number of platforms without the use of commercial kits.     

FSGO point charge models—Their accuracy and extension to higher gaessians

An FSGO model of ethane demonstrates how currently used point charge models can give the wrong sign for the potential. The Hall point charge potential on the other hand is asymptotically accurate.An extension of the Hall point charge model to higher Gaussians is demonstrated. This extended model potential is shown to be of comparable accuracy to that of the spherical Gaussian model.     

Allele-specific amplification and electrochemiluminescence method for single nucleotide polymorphism analysis

A new approach combined the specificity of allele-specific amplification (ASA) with the sensitivity of electrochemilu- minescence (ECL) assay for single nucleotide polymorphism (SNP) analysis was proposed. Briefly, target gene was amplified by a biotin-labeled allele-specific forward primer and a Ru(bpy)32 (TBR)-labeled universal reverse primer. Then, the amplicon was captured onto streptavidin-coated paramagnetic beads through biotin label, and detected by measuring the ECL signal of TBR label. Different genotypes were distinguished according to the ECL values of the amplicons by different genotypic primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experiment results show that the different genotypes can be clearly distinguished by ASA–ECL assay. The method is useful in SNP analysis due to its sensitivity, safety, and simplicity.     

Electrochemical analysis of single nucleotide polymorphisms of p53 gene

Single nucleotide polymorphisms (SNPs) of cancer repression gene p53 were analyzed electrochemically with ferrocenyl naphthalene diimide (1) as a hybridization indicator. The SNPs studied were the transition to A from G in the codon for amino acid at positions 175, 248 or 273 and the transversion to C from G in the codon for the amino acid at position 72. Thus, 20-meric oligonucleotides carrying the SNP site were used both as a sample and a probe with the latter immobilized on an electrode. Even one base difference on the p53 gene resulted in a significant difference in the current response of 1 and the magnitude of the response correlated with the amount of the DNA hybrid on the electrode. Moreover, when PCR products of exon 4, on which the P72/R72 SNP resides, of the p53 gene were analyzed by this method, the heterozygote and homozygotes were discriminated with modest precision.     

Recognition of single nucleotide polymorphisms using scanning potential hairpin denaturation

Conventional single nucleotide polymorphism (SNP) assays, which based their detection on the stringency or temperature of the washing buffers, have encountered difficulties to distinguish a single base pair mismatch from a perfect match. In this study, scanning potential hairpin denaturation (SPHD) has been developed to detect SNP in a sensitive and reliable manner. Combined with hairpin oligonucleotide probes, scanning surface electric potential was used to induce a dissociation of double-stranded DNA around a unique "melting potential" (Vm), and it generated a high-contrast SNP recognition signal. A 21 base pair p53 gene segment was used to test this novel method. A single nucleotide mismatch to the hairpin probes caused an average of 400-800 mV difference in melting potential against the perfect match, while the error of this assay was lower than 20 mV. Experiments demonstrated that the hairpin stem was critical to the method. The concept of scanning potential hairpin denaturation could also be used extensively in different areas of nucleotide hybridization based assays.     

Pinched flow fractionation devices for detection of single nucleotide polymorphisms

We demonstrate a new and flexible microfluidic based method for genotyping single nucleotide polymorphisms (SNPs). The method relies on size separation of selectively hybridized polystyrene microspheres in a microfluidic pinched flow fractionation (PFF) device. The microfluidic PFF devices with 13 mum deep channels were fabricated by thermal nanoimprint lithography (NIL) in a thin film of cyclic-olefin copolymer (mr-I T85) on a silicon wafer substrate, and the channels were sealed by thermal polymer bonding. Streptavidin coated polystyrene microspheres with a mean diameter of 3.09 microm and 5.6 microm were functionalized with biotin-labeled oligonucleotides for the detection of a mutant (Mt) or wild-type (Wt) DNA sequence in the HBB gene, respectively. Hybridization to functionalized beads was performed with fluorescent targets comprising synthetic DNA oligonucleotides or amplified RNA, synthesized using human DNA samples from individuals with point mutations in the HBB gene. Following a stringent wash, the beads were separated in a PFF device and the fluorescent signal from the beads was analyzed. Patients being wildtypes, heterozygotes or mutated respectively for the investigated mutation could reliably be diagnosed in the PFF device. This indicates that the PFF technique can be used for accurate and fast genotyping of SNPs.     

Fluorescence and visual detection of single nucleotide polymorphism using cationic conjugated polyelectrolyte

We report a simple assay for visual detection of single nucleotide polymorphisms (SNPs) with good sensitivity and selectivity. The selectivity is determined by Escherichia coli (E. coli) DNA ligase mediated circular formation upon recognition of the point mutation on DNA targets. Rolling cycle amplification (RCA) of the perfect-matched DNA target is then initiated using the in situ formed circular template in the presence of Phi29 enzyme. Due to amplification of the DNA target, the RCA product has a tandem-repeated sequence, which is significantly longer than that for the SNP strand. Direct addition of a cationic conjugated polymer of poly[9,9'-bis(6'-(N,N,N-trimethylammonium)hexyl)fluorene-co-9,9'-bis(2-(2-(2-(N,N,N-trimethylammonium)ethoxyl)-ethoxy)-ethyl)fluorene tetrabromide] containing 20 mol% 2,1,3-benzothiadiazole (PFBT(20)) into the RCA solution leads to blue-whitish fluorescent color for SNP strand and yellowish fluorescent color for amplified DNA, due to PFBT(20)/DNA complexation induced intrachain/interchain energy transfer. To further improve the contrast for visual detection, FAM-labeled peptide nucleic acid (PNA) was hybridized to each amplified sequence, which is followed by the addition of poly{2,7-[9,9-bis(6'-N,N,N-trimethylammoniumhexyl)]fluorene-co-2,5-difluoro-1,4-phenylene dibromide} (PFP). The PNA/DNA hybridization brings PFP and FAM-PNA into close proximity for energy transfer, and the solution fluorescent color appears green in the presence of target DNA with a detection limit of 1 nM, which is significantly improved as compared to that for most reported visual SNP assay.     

Surface-enhanced Raman scattering detection of bacteria on microarrays at single cell levels using silver nanoparticles

We describe a method for the synthesis of SERS-active silver nanoparticles (AgNPs) directly on the surface of bacteria (bacteria@AgNPs), specifically of E. coli cells. This straightforward strategy allows for the sensitive determination of bacteria on a microarray platform. Antibodies were used as selective receptors on the microarray surface. The Raman signal of bacteria@AgNPs is about 10 times higher than that obtained previously with microarrays based on mixing bacteria and AgNPs (bacteria+AgNPs). The optimum SERS enhancement of bacteria@AgNPs is obtained under 633-nm laser excitation, and this most likely is due to the plasmonic interaction of aggregated AgNPs. The method allows for an identification and quantification even of single E. coli bacteria. In our perception, this straightforward approach represents a most valuable tool for the detection of E. coli and, conceivably, of other bacteria, and thus has a large potential in environmental monitoring, medical diagnosis, and in food safety and quality control.

Synthesizing AgNPs directly on the surface of bacteria is demonstrated to be a highly efficient approach for a label-free readout of bacteria microarrays by surface-enhanced Raman scattering (SERS), resulting in signals about 10 times higher than previously reported results.

     

Homogeneous and label-free bioluminescence detection of single nucleotide polymorphism with rolling circle amplification

Integration of rolling circle amplification and sensitive bioluminescent detection of pyrophosphate, a homogeneous and label-free method has been developed for detecting single nucleotide polymorphism.     

Imperfect units of an extended microsatellite structure involving single nucleotide changes

Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) are widely used as markers in human genome studies. We have characterized a highly polymorphic STR locus (D20S85) with (AAAG)n repeats, by a combination of direct DNA sequencing and single-strand conformation polymorphism (SSCP) analysis. Eight STR alleles were first identified on denaturing gels, and SSCP gels were then used to demonstrate the existence of previously indistinguishable multiple alleles at the locus on the basis of variable allelic flanking sequences. This was confirmed by direct sequencing of the alleles. Four transitions, two G to A and two A to G in the 5'-flanking region of the locus at positions 14, 22, 24, and 26 effectively subdivided the STR alleles into two groups, with frequencies of 0.431 and 0.569, respectively. The mutational processes that generated the polymorphisms involved both simple changes in the number of AAAG repeats and single nucleotide mutations in the region flanking the repeat. The findings have potential application in the avoidance of false linkage and association. A composite locus of this nature, with separate STR and SNP evolutionary histories and resulting from different mutational processes, also could have wide application in studies of selection, drift, migration and inbreeding.