Single Nucleotide Polymorphism Genotyping Method Research Articles

A direct PCR–based SNP marker–assisted selection system (D-MAS) for different crops

As more and more re-sequencing genome data in crops were released, SNPs (single nucleotide polymorphisms) are easily achieved for genotyping and show the highest abundance among all kinds of molecular markers. However, high-throughput SNP genotyping methods, such as KASP (Kompetitive Allele Specific PCR), TaqMan, or ARMS (amplification refractory mutation system), are always labor on DNA extraction and depending on expensive equipment. Therefore, most breeders are undergoing the bottleneck of lacking an easy, cost-saving, and stable genotyping system. Here, we report a direct PCR–based medium-throughput SNP marker–assisted selection (D-MAS) system suits available major crops including rice, wheat, maize, and rapeseed. The D-MAS system, which reduces the time of manual operation and result analysis dramatically, contains (1) seedling breeding in greenhouse; (2) high-throughput DNA extraction by alkaline lysis; and (3) gel-free SNP marker detection with 384-well by PARMS (penta-primer amplification refractory mutation) or KASP genotyping system. The stability of alkaline lysis DNA was validated by flexible dilution fold and long storing time under low temperatures. The alkaline lysis DNA from four F2 populations of rice, wheat, maize, and rapeseed showed equal efficiency in SNP calling relative to the ones from cetyl trimethylammonium bromide (CTAB) method. With the alkaline lysis DNA, PAMRS showed denser genotype cluster than KASP. Furthermore, the D-MAS system was adaptive with rice old leaves. The throughput and efficiency of the D-MAS system were validated in the hybrid rice seed purity test with a Xian-Geng-specific SNP marker which is also recommended to select ipa1-2d gene in the rice molecular breeding. In conclusion, we proposed a direct PCR–based SNP calling pipeline, which could be a simple, cheap, and robust standard operation procedure (SOP) of molecular breeding for different crops and get extensive use in most laboratory.

Trp2027Cys mutation evolves in Digitaria insularis with cross-resistance to ACCase inhibitors

Sourgrass (Digitaria insularis) is one of the most problematic weeds in South America because glyphosate resistance is widespread across most crop production regions. Acetyl coenzyme A carboxylase (ACCase)-inhibiting herbicides have been intensively used to manage D. insularis, which substantially increased selection pressure for this class of herbicides. We confirmed resistance to ACCase herbicides in a D. insularis population from Brazil and characterized its molecular basis. Resistant plants showed high level of resistance to haloxyfop (resistance factor, RF = 613-fold), low level of resistance to pinoxaden (RF = 3.6-fold), and no resistance to clethodim. A target-site mutation, Trp2027Cys, was found in the ACCase sequence from resistant plants. A protein homology model shows that the Trp2027Cys mutation is near the herbicide-binding pocket formed between two ACCase chains, and is predicted to obstruct the access of aryloxyphenoxypropionates (FOP) herbicides to the binding site. A qPCR-based single nucleotide polymorphism genotyping method was validated to discriminate susceptible (wild-type Trp2027) and resistant (mutant Cys2027) alleles. All resistant plants were homozygous for the mutation and the assay could be used for early detection of resistance in D. insularis field samples with suspected resistance to ACCase inhibitors.

Construction of a rapid microfluidic-based SNP genotyping (MSG) chip for ancestry inference

In forensics, ancestry inference can provide leads for criminal investigation. Therefore, the time needed to generate results is the first priority. Single nucleotide polymorphisms (SNPs) are widely used genetic markers for ancestry inference. In this study, a rapid microfluidic-based SNP genotyping (MSG) chip was established to detect 72 autosomal SNPs for ancestry inference of East Asian populations. The DNA template was infused into the chip and distributed into reaction chambers with pre-spotted primer pairs under centrifugation. After sealing the inlets/outlets and the connections among adjacent reaction chambers, the chip was placed onto a plate thermocycler for competitive allele-specific PCR. The SNP genotyping results were generated by analyzing the extracted fluorescence signal of each reaction chamber after PCR was completed. The detection process took less time (2.5 h) and was simpler than other SNP genotyping methods, such as SNaPshot and MassArray. To assess the performance of the chip, its accuracy, specificity, and sensitivity were evaluated. A total of 50 samples were genotyped using the chip, and the consistency of all of the typing and sequencing results was 100%. For ancestry inference of unknown individuals, a reference database of 3628 individuals from 57 populations was employed, and the ancestry origin of 110 test samples was inferred, demonstrating that the differentiation of East Asian subpopulations can be achieved through the MSG chip method. Overall, the MSG chip method established in this study can effectively be used for rapid and accurate ancestry inference.

Comparison of Methods to Distinguish Diploid and Tetraploid Potato in Applied Diploid Breeding

Diploid (2n = 2x = 24) and tetraploid (2n = 4x = 48) germplasm are commonly used in potato breeding programs. Potato breeders need to efficiently and inexpensively differentiate between diploid and tetraploid progeny in dihaploid induction crosses as well as 2x-2x crosses where 2n gametes may occur in the parents. In this study, we compared the chloroplast count, genome-wide SNP genotyping and flow cytometry methods to determine ploidy. Twenty-eight clones were used as reference samples (14 diploid lines (2n = 2x = 24), 14 tetraploid (2n = 4x = 48) varieties and advanced breeding lines) to compare the three ploidy determination methods. The chloroplast count method was used to determine the ploidy level in the 28 reference samples, and 102 potato breeding lines derived from diploid (2x-2x) crosses. The Infinium 12 K V2 Potato Single Nucleotide Polymorphism (SNP) Array was used to examine the 28 reference samples and the 102 breeding lines. The results obtained from both chloroplasts counts and SNP genotyping techniques determined that there was a total of 84 diploid lines and 18 tetraploid lines. Flow cytometry was also used to determine ploidy level in a subset of 42 lines (28 reference lines set and 14 breeding lines). All three methods of ploidy determination (chloroplast counting, SNP genotyping and flow cytometry) agreed for all samples evaluated. These results demonstrate the usefulness of chloroplast counting as an efficient and inexpensive method for breeders to differentiate ploidy between diploid and tetraploid potato in applied breeding programs.

Genome sequencing and conservation genomics in the Scandinavian wolverine population.

Genetic approaches have proved valuable to the study and conservation of endangered populations, especially for monitoring programs, and there is potential for further developments in this direction by extending analyses to the genomic level. We assembled the genome of the wolverine (Gulo gulo), a mustelid that in Scandinavia has recently recovered from a significant population decline, and obtained a 2.42Gb draft sequence representing >85% of the genome and including >21,000protein-coding genes. We then performed whole-genome resequencing of 10Scandinavian wolverines for population genomic and demographic analyses. Genetic diversity was among the lowest detected in a red-listed population (mean genome-wide nucleotide diversity of 0.05%). Results of the demographic analyses indicated a long-term decline of the effective population size (Ne ) from 10,000well before the last glaciation to <500 after this period. Current Ne appeared even lower. The genome-wide FIS level was 0.089 (possibly signaling inbreeding), but this effect was not observed when analyzing a set of highly variable SNP markers, illustrating that such markers can give a biased picture of the overall character of genetic diversity. We found significant population structure, which has implications for population connectivity and conservation. We used an integrated microfluidic circuit chip technology to develop an SNP-array consisting of 96 highly informative markers that, together with a multiplex pre-amplification step, was successfully applied to low-quality DNA from scat samples. Our findings will inform management, conservation, and genetic monitoring of wolverines and serve as a genomic roadmap that can be applied to other endangered species. The approach used here can be generally utilized in other systems, but we acknowledge the trade-off between investing in genomic resources and direct conservation actions.

High-throughput blood sample preparation for single nucleotide polymorphism genotyping in less than 25 min

Straightforward, rapid and high-throughput pretreatment for single nucleotide polymorphisms (SNP) genotyping is critically needed in clinical practice. However, all existing SNP genotyping methods require DNA purification step, which is labor-intensive and time-consuming. We develop a protocol for SNP genotyping by combining whole blood lysis (WBL) with qPCR and justify the practicality of our method in blood samples from 140 donors, including 40 samples from healthy donors, and 100 samples from donors with either low white blood cell counts, high level of serum uric acid or triglyceride. When compared with Sanger sequencing, the gold standard for SNP genotyping, our method exhibits a 100% consistency in the aspect of sensitivity and specificity. In addition, our method can obtain amplifiable DNA within 25 mins (which is the fastest to the best of our knowledge) from 48 samples. The blood samples, even with low white blood cell counts, high level of serum uric acid or triglyceride could not affect the sensitivity and specificity of our method. Our study demonstrates that the combination of WBL and qPCR genotyping can serve as a high-throughput and robust approach for routine clinical SNP genotyping.

Toward fast and accurate SNP genotyping from whole genome sequencing data for bedside diagnostics.

Genotyping a set of variants from a database is an important step for identifying known genetic traits and disease-related variants within an individual. The growing size of variant databases as well as the high depth of sequencing data poses an efficiency challenge. In clinical applications, where time is crucial, alignment-based methods are often not fast enough. To fill the gap, Shajii et al. propose LAVA, an alignment-free genotyping method which is able to more quickly genotype single nucleotide polymorphisms (SNPs); however, there remains large room for improvements in running time and accuracy. We present the VarGeno method for SNP genotyping from Illumina whole genome sequencing data. VarGeno builds upon LAVA by improving the speed of k-mer querying as well as the accuracy of the genotyping strategy. We evaluate VarGeno on several read datasets using different genotyping SNP lists. VarGeno performs 7-13 times faster than LAVA with similar memory usage, while improving accuracy. VarGeno is freely available at: https://github.com/medvedevgroup/vargeno. Supplementary data are available at Bioinformatics online.

Comparison of three PCR-based assays for SNP genotyping in plants

BackgroundPCR allelic discrimination technologies have broad applications in the detection of single nucleotide polymorphisms (SNPs) in genetics and genomics. The use of fluorescence-tagged probes is the leading method for targeted SNP detection, but assay costs and error rates could be improved to increase genotyping efficiency. A new assay, rhAmp, based on RNase H2-dependent PCR (rhPCR) combined with a universal reporter system attempts to reduce error rates from primer/primer and primer/probe dimers while lowering costs compared to existing technologies. Before rhAmp can be widely adopted, more experimentation is required to validate its effectiveness versus established methods.ResultsThe aim of this study was to compare the accuracy, sensitivity and costs of TaqMan, KASP, and rhAmp SNP genotyping methods in sugar beet (Beta vulgaris L.). For each approach, assays were designed to genotype 33 SNPs in a set of 96 sugar beet individuals obtained from 12 parental lines. The assay sensitivity was tested using a series of dilutions from 100 to 0.1 ng per PCR reaction. PCR was carried out on the QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific, USA). The call-rate, defined as the percentage of genotype calls relative to the possible number of calls, was 97.0, 97.6, and 98.1% for TaqMan, KASP, and rhAmp, respectively. For rhAmp SNP, 24 of the 33 SNPs demonstrated 100% concordance with other two technologies. The genotype concordance with either technologies for the other 9 targets was above 99% (99.34–99.89%).ConclusionThe sensitivity test demonstrated that TaqMan and rhAmp were able to successfully determine SNP genotypes using as little as 0.2 ng DNA per reaction, while the KASP was unable to ascertain SNP states below 0.9 ng of DNA per reaction. Comparative cost per reaction was also analyzed with rhAmp SNP offering the lowest cost per reaction. In conclusion, rhAmp produced more calls than either TaqMan or KASP, higher signal to NTC data while offering the lowest cost per reaction.

HRM and SNaPshot as alternative forensic SNP genotyping methods

Single nucleotide polymorphisms (SNPs) have been widely used in forensics for prediction of identity, biogeographical ancestry (BGA) and externally visible characteristics (EVCs). Single base extension (SBE) assays, most notably SNaPshot® (Thermo Fisher Scientific), are commonly used for forensic SNP genotyping as they can be employed on standard instrumentation in forensic laboratories (e.g. capillary electrophoresis). High resolution melt (HRM) analysis is an alternative method and is a simple, fast, single tube assay for low throughput SNP typing. This study compares HRM and SNaPshot®. HRM produced reproducible and concordant genotypes at 500 pg, however, difficulties were encountered when genotyping SNPs with high GC content in flanking regions and differentiating variants of symmetrical SNPs. SNaPshot® was reproducible at 100 pg and is less dependent on SNP choice. HRM has a shorter processing time in comparison to SNaPshot®, avoids post PCR contamination risk and has potential as a screening tool for many forensic applications.

The effects of interleukin-28B gene polymorphisms on antiviral treatment response in patients with chronic hepatitis C

Objective To investigate the relationship between interleukin (IL)-28B gene polymorphisms (rs12979860 and rs8099917) and treatment response in patients with chronic hepatitis C in China. Methods Taqman probes single nucleotide polymorphism genotyping methods were used to detect the genotypes of rs12979860 (C/T) and rs8099917 (T/G) located at IL-28B gene in 105 included patients. The patients were treated with standard doses of pegylated interferon plus ribavirin and were followed up regularly for therapeutic response and adverse reaction. The relationship between IL-28B gene polymorphism and antiviral treatment response of patients were analyzed. Categorical data were analyzed using Pearson chi-square test or Fisher exact test. Results Totally 105 cases were included in our study and 2 cases lost to follow-up because of moving away. Eight-one cases (78.6%) of the remaining 103 patients were CC/TT genotype (CC/TT group) at rs12979860 and rs8099917, 19 cases (18.4%) were CT/TG (CT/TG group) and 3 cases (2.9%)were TT/TG (TT/TG group). No other genotypes were detected and linkage disequilibrium was discovered at the two polymorphism loci (r2=0.11). After 4 weeks of treatment, 35 cases (43.2%) in CC/TT group, 3 cases (15.8%) in CT/TG group and non in TT/TG group achieved rapid virological response (RVR). There were statistically significant differences among three groups (P=0.033). After 12 weeks of treatment, 45 cases (55.6%) in CC/TT group, 6 cases (31.6%) in CT/TG group and none in TT/TG group achieved early virological response (EVR). There were statistically significant differences among three groups (P=0.025). At the end of the treatment, 68 cases (83.9%) in CC/TT group, 10 cases (52.6%) in CT/TG group and only 1 case (33.3%) in TT/TG group achieved end-of-treatment response (ETR). There were significant statistical differences among the three groups (P=0.003). After 24 weeks of follow-up, 62 cases (76.5%) in CC/TT group, 9 cases (47.4%) in CT/TG group and 1 case (33.3%) in TT/TG group achieved sustained virological response (SVR). There were statistically significant differences among the three groups (P=0.014). One hundred and one cases in CC/TT group developed adverse events, among them 19 cases needed clinical treatment. There were 43 cases in CT/TG group developed adverse events and 9 cases needed treatment. Seven cases in TT/TG group developed adverse events and only 1 case needed treatment. There were no statistically significant difference among three groups (χ2=0.139, P>0.05). Conclusions The genotype of rs12979860 (C/T) and rs8099917 (T/G) at IL-28B gene could affect the treatment response in patients with chronic hepatitis C. RVR and SVR are higher in patients with genotype CC/TT, which might help to guide HCV treatment. Key words: Interleukin-28B; Hepatitis C, chronic; Polymorphism, single nucleotide; Antiviral response

A DNAzyme sensor based on target-catalyzed hairpin assembly for enzyme-free and non-label single nucleotide polymorphism genotyping

Single nucleotide polymorphisms (SNPs) are widely existed in human genome and associated with many diseases. Traditional PCR-based methods for SNP genotyping require protein enzyme, precise control of temperature and removal of resultant products, making the whole process labor intensive and time consuming. Although G-quadruplex DNAzyme-based assays provide many advantages over traditional approaches, the relatively low catalytic activity of DNAzyme becomes an unfavorable factor in its application process. Therefore, amplification of DNAzyme for further determination is of great desire in bioanalysis. In this work, we have developed an enzyme-free and non-label DNAzyme sensor for SNP genotyping based on target-catalyzed hairpin assembly (CHA) for DNAzyme amplification. The proposed sensor, carried out on microfluidic chemiluminescence (CL) assay, can sensitively discriminate rs242557 hotspot-SNP, the A/G single-nucleotide variation on human chromosome associated with Alzheimer's disease, with an absolute detection limit of 0.3 fmol.

An innovative SNP genotyping method adapting to multiple platforms and throughputs.

An innovative genotyping method designated as semi-thermal asymmetric reverse PCR (STARP) was developed for genotyping individual SNPs with improved accuracy, flexible throughputs, low operational costs, and high platform compatibility. Multiplex chip-based technology for genome-scale genotyping of single nucleotide polymorphisms (SNPs) has made great progress in the past two decades. However, PCR-based genotyping of individual SNPs still remains problematic in accuracy, throughput, simplicity, and/or operational costs as well as the compatibility with multiple platforms. Here, we report a novel SNP genotyping method designated semi-thermal asymmetric reverse PCR (STARP). In this method, genotyping assay was performed under unique PCR conditions using two universal priming element-adjustable primers (PEA-primers) and one group of three locus-specific primers: two asymmetrically modified allele-specific primers (AMAS-primers) and their common reverse primer. The two AMAS-primers each were substituted one base in different positions at their 3' regions to significantly increase the amplification specificity of the two alleles and tailed at 5' ends to provide priming sites for PEA-primers. The two PEA-primers were developed for common use in all genotyping assays to stringently target the PCR fragments generated by the two AMAS-primers with similar PCR efficiencies and for flexible detection using either gel-free fluorescence signals or gel-based size separation. The state-of-the-art primer design and unique PCR conditions endowed STARP with all the major advantages of high accuracy, flexible throughputs, simple assay design, low operational costs, and platform compatibility. In addition to SNPs, STARP can also be employed in genotyping of indels (insertion-deletion polymorphisms). As vast variations in DNA sequences are being unearthed by many genome sequencing projects and genotyping by sequencing, STARP will have wide applications across all biological organisms in agriculture, medicine, and forensics.

Biogeography of Oenococcus oeni Reveals Distinctive but Nonspecific Populations in Wine-Producing Regions.

This study reports the isolation, genotyping, and geographic distribution analysis of the largest collection of O. oeni strains performed to date. It reveals that there is very high diversity of strains in each region, the majority of them being detected in a single region. The study also reports the development of an SNP genotyping method that is useful for analyzing the distribution of O. oeni phylogroups. The results show that strains are not genetically adapted to regions but to specific types of wines. They reveal new phylogroups of strains, particularly two phylogroups associated with white wines and red wines of Burgundy. Taken together, the results shed light on the diversity and specificity of wild strains of O. oeni, which is crucial for understanding their real contribution to the unique properties of wines.

Association between genetic variations of NMDA receptor NR3 subfamily genes and heroin addiction in male Han Chinese

Growing amounts of evidence suggest that N-Methyl-d-aspartate (NMDA) receptor mediated glutamate neurotransmission may be involved in the pathophysiology of drug dependence. The NMDA receptor consists of three subfamilies (NR1, NR2, and NR3). The ability of subunit NR3 to negatively modulate the NMDA receptor function makes it an attractive candidate gene of heroin addiction. The purpose of this study is to explore the association between four single nucleotide polymorphisms (SNPs) of NR3 gene and heroin addiction. Genotyping of two SNPs (rs3739722 and rs17189632) in GRIN3A and two SNPs (rs4807399 and rs2240158) in GRIN3B was performed using TaqMan SNP genotyping method. The association between heroin addiction and these SNPs was assessed among 332 male heroin dependent patients and 400 male normal control subjects. The results showed the genotype and allele frequencies of rs17189632 and rs2240158 were significantly different between the cases and the controls (nominal P values were 0.0284, 0.0136 for rs17189632; 0.0048, 0.0013 for rs2240158, respectively). After Bonferroni correction, rs2240158 of GRIN3B was still found to be associated with heroin addiction. The frequencies of haplotype C-A at GRIN3A (rs3739722-rs17189632) and of C-C and C-T at GRIN3B (rs4807399-rs2240158) differed significantly between the cases and the controls. The genotype and allele distributions of rs3739722 and rs4807399 were not significantly different between in the cases and in the controls (P>0.05). These results suggest that GRIN3A rs17189632 and GRIN3B rs2240158 may contribute to the susceptibility of heroin addiction.

Genetic variations of DICKKOPF family genes might not be associated with gastric cancer susceptibility.

BackgroundRecent studies have implicated that members of the DICKKOPF (DKK) were causally involved in large number of human cancers. This study was designed to investigate the relationship between the genetic variations of DKK family genes and the risk of gastric cancer (GC).MethodsSix SNPs (single nucleotide polymorphisms) of DKK family genes, including rs2241529 in DKK1, rs3733635, rs17037102 and rs419764 in DKK2, rs3206824 in DKK3 and rs2073664 in DKK4, were selected and genotyped by restriction fragment length polymorphism (RFLP) and TaqMan SNP genotyping methods in 409 GC cases and 554 cancer-free controls in the Han population in eastern China.ResultsNone of the six SNPs achieved significant association with the overall GC risk and stratified analysis by age, gender, smoking status, drinking status, tumor location and pathological classification confirmed these non-significant associations.ConclusionsOur study indicated that the studied six SNPs of DKKs would not be the risk factors for GC in this Han Chinese population. Studies of larger population for different ethnicities will be needed to warrant our findings.

Applying SNP-Derived Molecular Coancestry Estimates to Captive Breeding Programs.

Captive breeding programs for wildlife species typically rely on pedigrees to inform genetic management. Although pedigree-based breeding strategies are quite effective at retaining long-term genetic variation, management of zoo-based breeding programs continues to be hampered when pedigrees are poorly known. The objective of this study was to evaluate 2 options for generating single nucleotide polymorphism (SNP) data to resolve unknown relationships within captive breeding programs. We generated SNP data for a zoo-based population of addax (Addax nasomasculatus) using both the Illumina BovineHD BeadChip and double digest restriction site-associated DNA (ddRAD) sequencing. Our results demonstrated that estimates of allele sharing (AS) between pairs of individuals exhibited low variances. Average AS variances were highest when using 50 loci (SNPchipall = 0.00159; ddRADall = 0.0249), but fell below 0.0003 for the SNP chip dataset when sampling ≥250 loci and below 0.0025 for the ddRAD dataset when sampling ≥500 loci. Furthermore, the correlation between the SNPchipall and ddRADall AS datasets was 0.88 (95%CI = 0.84-0.91) when subsampling 500 loci. Collectively, our results indicated that both SNP genotyping methods produced sufficient data for accurately estimating relationships, even within an extremely bottlenecked population. Our results also suggested that analytic assumptions historically integrated into the addax pedigree are not adversely impacting long-term pedigree-based management; kinships calculated from the analytic pedigree were significantly correlated (P << 0.001) with AS estimates. Overall, our conclusions are intended to serve as both a proof of concept and a model for applying molecular data to the genetic management of captive breeding programs.

Designing, Optimization and Validation of Tetra Primer ARMS-PCR Protocol for Genotyping Single Nucleotide Polymorphism rs4731702 (C/T) of KLF14 Gene Associated with Type 2 Diabetes Mellitus: A Study in San Luis, Argentina

New, quick, and inexpensive methods for genotyping KLF14 gene polymorphism through Tetra Primer ARMS-PCR were developed in the present investigation. Single nucleotide polymorphism (SNP) genotyping needs to be attempted to establish association between the identified SNP with metabolic trait for identification of molecular markers that can be used to identify individuals at risk of developing Diabetes Mellitus Type 2. In the current study, we have successfully genotyped the SNP rs4731702 (C/T) in KLF14 gene. Tetra primer ARMS-PCR protocol was optimized and validated for this SNP with short turn-around time and costs. The optimized techniques were tested on 60 samples of controls and type 2 Diabetes Mellitus individuals. Samples with known genotypes for the described gene, previously tested in duplicate using the sequencing methods, were employed for validation of the assay. Upon validation, complete concordance was observed between the Tetra Primer ARMS-PCR assays and the sequencing results. These results highlight the ability of Tetra Primer ARMS-PCR in genotyping of SNP in KLF14 gene. Our results provide direct evidence that Tetra Primer ARMS-PCR is a rapid, reliable, and cost-effective method for SNP genotyping of KLF14 gene in type 2 Diabetes Mellitus individuals.

Colorimetric SNP genotyping utilizing colloidal stability of double-stranded DNA-functionalized gold nanoparticles having a dangling end

Event Abstract Back to Event Colorimetric SNP genotyping utilizing colloidal stability of double-stranded DNA-functionalized gold nanoparticles having a dangling end Yoshitsugu Akiyama1, 2, Hiroto Shikagawa1, Shota Shiraishi1, Guoqing Wang1, Naoki Kanayama1, Tohru Takarada1 and Mizuo Maeda1 1 RIKEN, Bioengineering Laboratory, Japan 2 Tokyo University of Science, Faculty of Industrial Science and Technology, Japan Introduction: Facile and reliable analytical methods to discriminate single nucleotide polymorphisms (SNPs) are of great importance for the realization of personalized medicine. We have already reported a colorimetric SNP genotyping method using salt-induced, non-crosslinking aggregation of double-stranded DNA-modified gold nanoparticles (dsDNA-GNPs)[1]. To develop a more reliable method, the present study exploited a large difference in colloidal stability of dsDNA-GNPs with and without a dangling end is described (Figure 1). Experimental: According to a reported procedure[1], GNP with a diameter of 15 nm was functionalized with 16-nucleotide (nt) ssDNA. The base sequence of ssDNA was designed to be complementary to an SNP typing primer. A chemically synthesized fragment (56 base pairs) of the cytochrome P450 2C19*2 gene including the SNP site (G→A) was used as a model target. Single base extension of the 16-nt typing primer for the SNP site was carried out with the model target, DNA polymerase, and each ddNTP. To this reaction solution was added ssDNA-GNP (final conc. 5 nM) and NaCl (final conc. 1 M). The as-prepared mixture was incubated for 10 min at room temperature to induce non-crosslinking aggregation. The color change attributed to a surface plasmon resonance shift was detected with the naked eye. Results and Discussion: When the single base extension reaction using the model target of a homozygous mutant (the SNP site: T) in the presence of ddATP, ddGTP, or ddCTP, the distinct color change from red to purple through non-crosslinking aggregation of dsDNA-GNP was observed (Figure 1, left). This is due to the formation of fully matched dsDNA between ssDNA on the GNP surface and the unextended 16-nt typing primer. On the other hand, it showed no color change (red) with ddTTP (Figure 1, right), because the dsDNA having a dangling end was formed on the surface with the single-base-extended 17-nt typing primer, leading to the nanoparticle’s dispersion by the entropic repulsion. In addition, the corresponding color changes were also obtained when using the model target of the homozygous wild-type gene, as well as that of the heterozygous gene[2]. Conclusion: Colorimetric SNP genotyping of the cytochrome P450 2C19*2 gene by unique colloidal stability of dsDNA-GNP with a dangling end was successfully demonstrated. It will be potentially achievable to develop a clinical sample analysis. This work was supported by a Grant-in-Aid for Scientific Research (S) (No. 25220204) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by a Grant for Molecular Systems Research provided by RIKEN.

A Potential Polymorphism in the Promoter of Let-7 is Associated With an Increased Risk of Intracranial Aneurysm: A Case-Control Study.

Let-7 family plays a key role in the progression of atherosclerosis and intracranial aneurysm (IA). We hypothesized that rs10877887 and rs13293512 polymorphisms in the promoters of let-7 family may be associated with the susceptibility of IA. We genotyped the 2 single nucleotide polymorphisms (SNPs) in 305 patients with IA and 401 healthy controls. The rs10877887 was analyzed using a polymerase chain reaction-restriction fragment length polymorphism assay, and the rs13293512 was analyzed using a TaqMan SNP genotyping method. The relative expression of let-7 family was measured in plasma of cases and controls using real-time PCR. We found that the rs13293512CT genotype was associated with a significantly increased risk of developing IA in a heterozygote comparison (adjusted OR = 1.43, 95% CI, 1.00–2.05, P = 0.048) and dominant comparison (adjusted OR = 1.44, 95% CI, 1.02–2.03, P = 0.04). Combined analysis showed that the rs10877887TT and rs13293512CC/CT genotypes had a significantly increased risk of IA (OR = 1.67, 95% CI, 1.04–2.68, P = 0.03). Moreover, the levels of let-7a, let-7d, and let-7f were downregulated in IA patients, and patients with the rs13293512CC/CT genotypes had a lower level of let-7a than those with rs13293512TT genotype (P = 0.03). These findings indicate that the rs13293512CC/CT is a risk factor for the development of IA, possibly because of the genotypes resulting in a lower level of let-7a.

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria.

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.