Exploring of tri-allelic SNPs using pyrosequencing and the SNaPshot methods for forensic application

Tri-allelic single nucleotide polymorphisms (SNPs) are potential forensic markers for DNA analysis. Currently, only a limited number of tri-allelic SNP loci have been proved to be fit for forensic application. In this study, we aimed to develop an effective method to select and genotype tri-allelic SNPs based on both Pyrosequencing (PSQ) and the SNaPshot methods. 50 candidate SNPs were chosen from NCBI's dbSNP database and were analyzed by PSQ. The results revealed that 20 SNPs were tri-allelic and were located on 16 autosomal chromosomes. Then 20 SNP loci were combined in one multiplex polymerase chain reaction to develop a single base extension (SBE)-based SNP-typing assay. A total of 100 unrelated Chinese individuals were genotyped by this assay and allele frequencies were estimated. The total discrimination power was 0.999999999975 and the cumulative probability of exclusion was 0.9937. These data demonstrated that the strategy is a rapid and effective method for seeking and typing tri-allelic SNPs. In addition, the 20 tri-allelic SNP multiplex typing assay may be used to supplement paternity testing and human identification.     

Label-free genotyping of single-nucleotide polymorphisms for DNA and RNA targets using a cationic polythiophene derivate

By using the specific primer extension reaction, a new assay for genotyping of single-nucleotide polymorphisms (SNPs) has been demonstrated. The assay relies on the conformational and colorimetric change of water-soluble polythiophene derivative, poly[3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydrochloride] (PMNT), upon forming interpolyelectrolyte complex with extended double strand DNA and non-extended single strand DNA. All three kinds of SNP genotypes can be colorimetrically identified with one primer extension reaction in homogeneous solution. Moreover, combining with the specific digestion of RNA strands in the RNA/DNA hybrids, the proposed assay can also be applied to SNP genotyping for RNA templates. The SNP genotyping assay does not require chemical modification of oligonucleotide probes and nucleic acid targets and any separation step. It would be useful for routinely SNP detection in ordinary laboratories.     

A SNaPshot assay for genotyping 44 individual identification single nucleotide polymorphisms

Single nucleotide polymorphisms (SNPs), which have relatively low mutation rates and can be genotyped after PCR with shorter amplicons compared with short tandem repeats (STRs), are being considered as potentially useful markers in forensic DNA analysis. Those SNPs with high heterozygosity and low Fst (F-statistics) in human populations are described as individual identification SNPs, which perform the same function as STRs used in forensic routine work. In the present study, we developed a multiplex typing method for analyzing 44 selected individual identification SNPs simultaneously by using multiplex PCR reaction in association with fluorescent labeled single base extension (SBE) technique. PCR primers were designed and the lengths of the amplicons ranged from 69 to 125?bp. The population genetics data of 79 unrelated Chinese individuals for the 44 SNP loci were investigated and a series of experiments were performed to validate the characteristic of the SNP multiplex typing assay, such as sensitivity, species specificity and the performance in paternity testing and analysis of highly degraded samples. The results showed that the 44-SNPs multiplex typing assay could be applied in forensic routine work and provide supplementary data when STRs analysis was partial or failed.     

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.     

Use of an electrochemically labeled nucleotide terminator for known point mutation analysis

A novel single nucleotide polymorphism (SNP) assay utilizing an electrochemically tagged chain terminator is described. The system employs the single-base extension (SBE) technique coupled to capillary gel electrophoresis with end-column electrochemical detection. A redox-labeled chain terminator, ferrocene-acycloATP, is used in the SBE reaction. When the mutation site corresponds to the labeled chain terminator, the extension product is rendered electroactive. The reaction mixture is subsequently separated by capillary gel electrophoresis and the extension product detected at the separation anode with sinusoidal voltammetry. This work demonstrates the first known SNP assay utilizing redox-active chain terminators coupled to electrochemical detection. The methodology presented could lead to a fast, simple, and cost-effective SNP scoring system.     

Analysis of four PCR/SNaPshot multiplex assays analyzing 52 SNPforID markers

The SNPfor ID consortium identified a panel of 52 SNPs for forensic analysis that has been used by several laboratories worldwide. The original analysis of the 52 SNPs was based on a single multiplex reaction followed by two single‐base‐extension (SBE) reactions each of which was analyzed using capillary electrophoresis. The SBE assays were designed for high throughput genetic analyzers and were difficult to use on the single capillary ABI PRISM 310 Genetic Analyzer and the latest generation 3500 Genetic Analyzer, as sensitivity on the 310 was low and separation of products on the 3500 with POP‐7™ was poor. We have modified the original assay and split it into four multiplex reactions, each followed by an SBE assay. These multiplex assays were analyzed using polymer POP‐4™ on ABI 310 PRISM® and polymers POP‐4™, POP‐6™ and POP‐7™ on the 3500 Genetic Analyzer. The assays were sensitive and reproducible with input DNA as low as 60 pg using both the ABI 310 and 3500. In addition, we found that POP‐6™ was most effective with the 3500, based on the parameters that we assessed, achieving better separation of the small SBE products; this conflicted with the recommended use of POP‐7™ by the instrument manufacturer. To support the use of the SNP panel in casework in Malaysia we have created an allele frequency database from 325 individuals, representing the major population groups within Malaysia. Population and forensic parameters were estimated for all populations and its efficacy evaluated using 51 forensic samples from challenging casework.     

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.     

Integrated platform for detection of DNA sequence variants using capillary array electrophoresis

We have developed a highly versatile platform that performs temperature gradient capillary electrophoresis (TGCE) for mutation/single-nucleotide polymorphism (SNP) detection, sequencing and mutation/SNP genotyping for identification of sequence variants on an automated 24-, 96- or 192-capillary array instrument. In the first mode, multiple DNA samples consisting of homoduplexes and heteroduplexes are separated by CE, during which a temperature gradient is applied that covers all possible temperatures of 50% melting equilibrium (Tms) for the samples. The differences in Tms result in separation of homoduplexes from heteroduplexes, thereby identifying the presence of DNA variants. The sequencing mode is then used to determine the exact location of the mutation/SNPs in the DNA variants. The first two modes allow the rapid identification of variants from the screening of a large number of samples. Only the variants need to be sequenced. The third mode utilizes multiplexed single-base extensions (SBEs) to survey mutations and SNPs at the known sites of DNA sequence. The TGCE approach combined with sequencing and SBE is fast and cost-effective for high-throughput mutation/SNP detection.     

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.     

Single nucleotide polymorphism analysis by allele-specific primer extension with real-time bioluminescence detection in a microfluidic device

A microfluidic approach for rapid bioluminescent real-time detection of single nucleotide polymorphism (SNP) is presented. The method is based on single-step primer extension using pyrosequencing chemistry to monitor nucleotide incorporations in real-time. The method takes advantage of the fact that the reaction kinetics differ between matched and mismatched primer-template configurations. We show here that monitoring the initial reaction in real time accurately scores SNPs by comparing the initial reaction kinetics between matched and mismatched configurations. Thus, no additional treatment is required to improve the sequence specificity of the extension, which has been the case for many allele-specific extension assays. The microfluidic approach was evaluated using four SNPs. Three of the SNPs included primer-template configurations that have been previously reported to be difficult to resolve by allele-specific primer extension. All SNPs investigated were successfully scored. Using the microfluidic device, the volume for the bioluminescent assay was reduced dramatically, thus offering a cost-effective and fast SNP analysis method.     

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.     

Microarray-based detection of Korean-specific <Emphasis Type="Italic">BRCA1</Emphasis> mutations

A reliable multiplex assay procedure to detect human genetic mutations in the breast cancer susceptibility gene BRCA1 using zip-code microarrays and single base extension (SBE) reactions is described. Multiplex PCR amplification was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in subsequent multiplex SBE reactions using bifunctional primers carrying a unique complementary zip sequence in addition to a mutation-site-specific sequence. The SBE primers, terminating one base before their mutation sites, were extended by a single base at a mutation site with a corresponding biotin-labeled ddNTP. Hybridization of the SBE products to zip-code microarrays was followed by staining with streptavidin–Cy3, leading to successful genotyping of several selected BRCA1 mutation sites with wild-type and heterozygote mutant samples from breast cancer patients. This work has led to the development of a reliable DNA microarray-based system for the diagnosis of human genetic mutations. Cheulhee Jung and Seong-Chun Yim contributed equally to this work.     

Improved adapter-ligation-mediated allele-specific amplification for multiplex genotyping by using software

Adapter-ligation-mediated allele-specific amplification (ALM-ASA) is a potential method for multiplex SNPs typing at an ultra low cost. Here, we describe a kind of software, which designs allele-specific primers for ALM-ASA assay on multiplex SNPs. DNA sequences containing SNPs of interest are submitted into the software which contains various endonucleases for options. Based on the SNP sequence information and the selected endonucleases, the software is capable of automatically generating sets of information needed to perform genotyping experiments. Each set contains a suitable endonuclease, qualified allele-specific primers with orientations and melting temperatures, sizes of allele-specific amplicons, and gel electropherograms simulated according to the sizes of the allele-specific amplicons and the mobility of DNA fragments in 2% agarose gel. Seven SNPs in the arylamines N-acetyltransferase 2 (NAT2) gene, five SNPs in the BRCA1 gene, five SNPs in the COMT gene, six SNPs in the CYP2E1 gene, five SNPs in the MPO gene, and six SNPs in the NRG1 gene were selected for evaluating the software. Without extra optimization, seven SNPs in the NAT2 gene were successfully genotyped for genomic DNA samples from 127 individuals by using the first set of allele-specific primers yielded by the software. Although several steps are used in the ALM-ASA assay, the whole genotyping process can be completed within 3 h by optimizing each step. Profiting from the software, the ALM-ASA assay is easy-to-perform, labor-saving, and accurate.     

A multiplex single nucleotide polymorphism genotyping method using ligase‐based mismatch discrimination and CE‐SSCP

Accuracy, simplicity, and cost‐effectiveness are the most important criteria for a genotyping method for SNPs compatible with clinical use. One method developed for SNP genotyping, ligase‐based discrimination, is considered the simplest for clinical diagnosis. However, multiplex assays using this method are limited by the detection method. Although CE has been introduced as an alternative to error prone microarray‐based detection, the design process and multiplex assay procedure are complicated because of the DNA size‐dependent separation principle. In this study, we developed a simple and accurate multiplex genotyping method using reaction condition‐optimized ligation and high‐resolution CE‐based SSCP. With this high‐resolution CE‐SSCP system, we are able to use similar‐sized probes, thereby eliminating the complex probe design step and simplifying the optimization process. We found that this method could accurately discriminate single‐base mismatches in SNPs of the tp53 gene, used as targets for multiplex detection.     

Highly multiplex and sensitive SNP genotyping method using a three‐color fluorescence‐labeled ligase detection reaction coupled with conformation‐sensitive CE

For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost‐effectiveness are the most important criteria. Among the methods currently being developed for SNP genotyping technology, the ligation‐dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error‐prone hybridization‐based detection, the multiplex assay process is complicated because of the size‐based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high‐resolution CE‐SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar‐sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three‐color fluorescence‐labeled probes.     

Expansion of a SNaPshot assay to a 55‐SNP multiplex: Assay enhancements,validation, and power in forensic science

A previously developed multiplex assay with 44 individual identification SNPs was expanded to a 55plex assay. Fifty‐four highly informative SNPs and an amelogenin sex marker were amplified in one PCR reaction and then detected with two SNaPshot reactions using CE. PCR primers for four loci, 28 single‐base extension primers, and the reaction conditions were altered to improve the robustness of the method. A detailed approach for allele calling was developed to guide analysis of the electropherogram. One hundred and eighty unrelated individuals and 100 father‐child‐mother trios of the Han population in Hebei, China were analyzed. No mutation was found in the SNP loci. The combined mean match probability and cumulative probability of exclusion were 1.327 × 10^?22 and 0.999932, respectively. Analysis of the 54 SNPs and 26 STRs (included in the AmpFLSTR Identifiler and Investigator HDplex kits) showed no significant linkage disequilibriums. Our research shows that the expanded SNP multiplex assay is an easily performed and valuable method to supplement STR analysis.     

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.     

一种基于磁性纳米粒子PCR的高通量SNP分型方法

利用磁性纳米粒子PCR扩增(MNPs-PCR)和等位基因特异性双色荧光探针(Cy3, Cy5)杂交, 建立了一种单核苷酸多态性(SNP)分型的新方法. 应用该方法对9个样本MTHFR基因的C677T多态进行检测, 野生和突变型样本正错配信号比大于9.0, 杂合型正错配信号比接近1.0, 分型结果经测序验证. 此方法无须产物纯化、浓缩, 扫描分型结果快速、直观, 是一种操作简单、快速、高通量、高灵敏度的分型方法.     

PCR-free MDR1 polymorphism identification by gold nanoparticle probes

Single nucleotide polymorphisms (SNPs) represent the most abundant source of genetic variation in the human genome, and they can be linked to genetic susceptibilities or varied pharmaceutical responses. Established SNP detection techniques are mainly PCR-based, which means that they involve complex, labor-intensive procedures, are easy contaminated, and can give false-positive results. Therefore, we have developed a simple and rapid MS-based disulfide barcode methodology that relies on magnifying the signal from a dual-modified gold nanoparticle. This approach permits direct SNP genotyping of total human genomic DNA without the need for primer-mediated enzymatic amplification. Disulfides that are attached to the gold nanoparticle serve as a “barcode” that allows different sequences to be discerned using MS detection. Specificity is based on two sequential oligonucleotide hybridizations, which include two steps: the first is the capture of the target by gene-specific probes immobilized onto magnetic beads; the second is the recognition of gold nanoparticles functionalized with allele-specific oligonucleotides. The sensitivity of this new method reaches down to the 0.1 fM range, thus approaching that of PCR. The feasability of this SNP identification methodology based on an MS-based disulfide barcode assay was demonstrated by applying it to genomic DNA samples representing all possible genotypes of the SNPs G2677T and C3435T in the human MDR1 gene. Due to its great advantage—the ability to perform SNP typing without the use of PCR—the assay was found to be simple, rapid and robust, and so may be highly suited to routine clinical detection as well as basic medical research.     

A new approach to detect a set of SNP-SNP markers: Combining ARMS-PCR with SNaPshot technology

Microhaplotypes are a new promising type of forensic genetic marker. Without the interference of stutter and high mutation rates as for STRs, and with short amplification lengths and a higher degree of polymorphism than single SNP, microhaplotypes composed of two SNPs, SNP–SNP, have a strong application potential. Currently, the most common method to detect microhaplotypes is massive parallel sequencing. However, the cost and extensive use of instruments limit its wide application in forensic laboratories. In this study, we screened 23 new SNP–SNP loci and established a new detection method by combining a multiplex amplification refractory mutation system-based PCR (ARMS-PCR) and SNaPshot technology based on CE. First, we introduced an additional deliberate mismatch at the antepenultimate base from the 3′ end of primers when designing ARMS-PCR for SNP 1 (the first SNP of the SNP–SNP). Then, single base extension primers for SNaPshot assay were designed next to the position of SNP 2 (the second SNP). Finally, 15 loci were successfully built into four panels and these loci showed a relatively high level of polymorphism in the Southwest Chinese Han population. All the loci had an average probability of informative genotypes (I value) of 0.319 and a combined discrimination power of 0.999999999. Therefore, this new detection system will provide a valuable supplement to current methods.