A multiplex assay with 52 single nucleotide polymorphisms for human identification

A total of 52 SNPs reported to be polymorphic in European, Asian and African populations were selected. Of these, 42 were from the distal regions of each autosome (except chromosome 19). Nearly all selected SNPs were located at least 100 kb distant from known genes and commonly used STRs. We established a highly sensitive and reproducible SNP-typing method with amplification of all 52 DNA fragments in one PCR reaction followed by detection of the SNPs with two single base extension reactions analysed using CE. The amplicons ranged from 59 to 115 bp in length. Complete SNP profiles were obtained from 500 pg DNA. The 52 loci were efficiently amplified from degraded samples where previously only partial STR profiles had been obtained. A total of 700 individuals from Denmark, Greenland, Somalia, Turkey, China, Germany, Taiwan, Thailand and Japan were typed, and the allele frequencies estimated. All 52 SNPs were polymorphic in the three major population groups. The mean match probability was at least 5.0 x 10(-19) in the populations studied. Typical paternity indices ranged from 336 000 in Asians to 549 000 in Europeans. Details of the 52 SNP loci and population data generated in this work are freely available at http://www.snpforid.org.     

Extension of the GOOD assay for genotyping single nucleotide polymorphisms by matrix-assisted laser desorption/ionization mass spectrometry

Over the past years several methods using mass spectrometry for high-throughput genotyping of single nucleotide polymorphisms (SNPs) have been developed. Most of these procedures require stringent purification. Only the GOOD assay does not need any sample purification. Here, several new implementations of this assay are presented. The molecular biological procedure of the GOOD assays is based on the principle that the analysis of DNA by matrix-assisted laser desorption/ionization (MALDI) is strongly dependent on the charge state. A 100-fold increase in sensitivity can be achieved if the analyzed DNA product is conditioned by a chemical procedure termed 'charge-tagging'. The GOOD assay starts with a PCR; allele-specific DNA molecules are generated by extension of modified primers. These contain up to three phosphorothioates and optionally a quaternary ammonium charged group with ddNTPs or alpha-S-ddNTPs. Then the unmodified part of the primers is digested by phosphodiesterase II and the negative charges of the phosphorothioates are neutralized by an alkylation reaction resulting in charge-tagged DNA products. Through the use of a novel DNA polymerase for the primer extension, which preferably incorporates ddNTPs over dNTPs, an enzymatic degradation of residual dNTPs from the PCR is not required. Additionally, the unique property of charge-tag technology is demonstrated to detect specifically on the same sample allele-specific DNA products carrying a positive charge-tag in the positive ion mode while products carrying a negative charge-tag are analyzed in the negative ion mode. We also generated zwitterionic allele-specific products that were detectable with high sensitivity in positive ion mode. The findings of this study raise interesting questions about the ionization process of nucleic acids in MALDI. The new variations of the GOOD assay were applied to genotype SNPs of a candidate gene for cardiovascular disease.     

Construction of an electrochemical DNA chip for simultaneous genotyping of single nucleotide polymorphisms

An electrochemical DNA chip was constructed for simultaneous genotyping of single nucleotide polymorphisms (SNPs) using genomic DNA extracted from blood samples. This chip consisted of electrodes located on a single piece of substrate and allele-specific oligonucleotide probes on the electrodes. As a first application, the 4 SNPs (MxA[-88], MxA[-123], MBL[X/Y], and MBL[A/B]), which have association with the efficacy of interferon therapy for HCV patient, were genotyped on the new DNA chip. Following hybridization of PCR products containing the 4 types of fragments, washing, bisbenzimide H33258 (Hoechst 33258) reaction and electrochemical analyses, 59 blood samples were genotyped by the chip method simultaneously. All procedures were completed within 2 h and the results were 100% concordant with those by the direct sequence method. The electrochemical DNA chip is expected to be a practical tool for SNPs genotyping.     

Electrochemical single nucleotide polymorphisms genotyping on surface immobilized three-dimensional branched DNA nanostructure

An electrochemical assay for single nucleotide polymorphisms (SNPs) genotyping is reported. Although electrochemical method is sensitive for DNA detection on surfaces, the ability of surface assay to precisely recognize DNA hybridization event is sacrificed to some extent due to the crowded confined surfaces environments that disfavor DNA hybridization. In the present study, we employed branched tetrahedron structure probes (TSPs) to replace regular linear single stranded DNA capture probes that were immobi...     

Fluorescence polarization for single nucleotide polymorphism genotyping

Single nucleotide polymorphisms (SNPs) are the most abundant variations in the human genome and have become the primary markers for genetic studies for mapping and identifying susceptible genes for complex diseases. Methods that genotype SNPs quickly and economically are of high values for these studies because they require a large amount of genotyping. Fluorescence polarization (FP) is a robust technique that can detect products without separation and purification and it has been applied for SNP genotyping. In this article the applications of FP in SNP genotyping are reviewed and one of the methods, the FP-TDI assay, is discussed in details. It is hoped that readers could get useful information for the applications of FP in SNP genotyping and some insights of the FP-TDI assay.     

Robust electrochemical system for screening single nucleotide polymorphisms

A highly sensitive and selective electrochemical DNA signaling scheme, which identifies the point mutation existing in target DNA sequence, is developed based on the combination of label-free hairpin probe (HP)/DNA endonuclease with zirconia (ZrO(2)) nanoparticle film, representing a promising screening platform for the accurate diagnosis of infections and genetic diseases as well as for environmental and forensic applications.     

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.     

Cost-effective interrogation of single nucleotide polymorphisms using the mismatch amplification mutation assay and capillary electrophoresis

The ability to characterize SNPs is an important aspect of many clinical diagnostic, genetic and evolutionary studies. Here, we designed a multiplexed SNP genotyping method to survey a large number of phylogenetically informative SNPs within the genome of the bacterium Bacillus anthracis. This novel method, CE universal tail mismatch amplification mutation assay (CUMA), allows for PCR multiplexing and automatic scoring of SNP genotypes, thus providing a rapid, economical and higher throughput alternative to more expensive SNP genotyping techniques. CUMA delivered accurate B. anthracis SNP genotyping results and, when multiplexed, saved reagent costs by more than 80% compared with TaqMan real-time PCR. When real-time PCR technology and instrumentation is unavailable or the reagents are cost-prohibitive, CUMA is a powerful alternative for SNP genotyping.     

A novel technique for detecting single nucleotide polymorphisms by analyzing consumed allele-specific primers

We present a simple and rapid polymerase chain reaction (PCR)-based technique, termed consumed allele-specific primer analysis (CASPA), as a new strategy for single nucleotide polymorphism (SNP) analysis. The method involves the use of labeled allele-specific primers, differing in length, with several noncomplementary nucleotides added in the 5'-terminal region. After PCR amplification, the amounts of the remaining primers not incorporated into the PCR products are determined. Thus, nucleotide substitutions are identified by measuring the consumption of primers. In this study, the CASPA method was successfully applied to ABO genotyping. In the present method, the allele-specific primer only anneals with the target polymorphic site on the DNA, so it is not necessary to analyze the PCR products. Therefore, this method is only little affected by modification of the PCR products. The CASPA method is expected to be a useful tool for typing of SNPs.     

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.     

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.     

Ligase chain reaction amplification for sensitive electrochemiluminescent detection of single nucleotide polymorphisms

Single nucleotide polymorphisms are the most common type of genetic variations among human beings and can serve as biomarkers for various types of diseases. In this work, based on ligase chain reaction amplification for the production of massive hemin/G-quadruplex DNAzymes to quench the electrochemiluminescent (ECL) emission of quantum dots (QDs), a universal and sensitive single nucleotide polymorphism detection method is described. During the ligase chain reaction process, the mutant K-ras target gene is recycled and exponentially duplicated, leading to the attachment of numerous G-rich sequences on the QD-embedded sensing surface. Upon the addition of the assistant sequences and hemin, numerous hemin/G-quadruplex DNAzymes are formed, which consume the dissolved oxygen in the detection buffer and result in significant quenching of QD ECL emission for sensitive single nucleotide polymorphism determination. The developed method shows a linear range of 50 fM to 50 pM and an estimated detection limit of 45 fM for the mutant K-ras gene. The proposed strategy also exhibits high selectivity towards the mutant K-ras gene against the co-existence of 10³-fold excess of the wild-type K-ras gene, which makes our method a useful addition to the alternatives for single nucleotide polymorphism monitoring.     

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.     

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.     

Quantitative genotyping of single nucleotide polymorphism by single-molecule multi-color fluorescence resonance energy transfer

We developed a new single nucleotide polymorphism (SNP) genotyping method based on single-molecule multi-color fluorescence resonance energy transfer (FRET). We demonstrated that this new method uses less than 1 fmol of sample and is also highly quantitative with a detection level of 1% or lower in the minor allele fraction.     

Detection of single nucleotide polymorphisms using electrospray ionization mass spectrometry: validation of a one-well assay and quantitative pooling studies

Single nucleotide polymorphisms (SNPs) are currently being mapped and databased at a remarkable pace, providing a viable means for understanding disease susceptibility, differential drug response and human evolution. Consequently, there is an increasing demand for SNP genotyping technologies that are simple, rapid, cost effective and readily amenable to automation for high-throughput analyses. In this study, we improved the Survivor Assay, a SNP detection method based on electrospray ionization mass spectrometry (ESI-MS), with several developments. One improvement is the development of a one-well assay, requiring no off-line purification of the polymerase chain reaction product, achieved by simple addition of reagent solution into a single well. Another is the on-line separation of magnesium and dideoxynucleotides using an in-house made monolithic metal chelating column, eliminating any off-line sample preparation prior to mass spectrometric analysis. Here the Survivor Assay is extended from a proof-of-principle concept to a validated method by genotyping six SNPs from five different regions of human genomic DNA in 55 individual samples with 100% accuracy. This improved Survivor Assay eliminates the tedious and time-consuming steps of sample preparation, minimizes sample handing and offers a high-throughput analysis of SNPs by ESI-MS. The current combined preparation and analysis time is 2 min per sample. The simplicity of this method has potential for full automation and parallel chromatography and, thus, reduced analysis time. In addition, we have adapted the Survivor Assay for quantitative SNP analysis in pooled DNA samples. The capabilities and sensitivity of this approach were evaluated. We demonstrate that an allele occurring at a frequency of 2% can consistently be quantitated.