Multiple sample amplification and genotyping integrated on a single electronic microarray.

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

Ensuring the genetic homogeneity of the mice used in laboratory experiments contributes to the Reduction aspect of the Three Rs, by maximising the quality of the data obtained from any animals that are used for these purposes, and ultimately reducing the numbers of animals used. Single nucleotide polymorphism (SNP) genotyping is especially suitable for use in the analysis of the genetic purity of model organisms such as the mouse, because bi-allelic markers remain fully informative when used to characterise crosses between inbred strains. Here, we attempted to apply a microarray-based method for a SNP marker to monitor the genetic quality of inbred mouse strains, so as to validate the reliability, stability and applicability of this SNP genotyping panel. The amplified PCR products containing four different SNP loci from four inbred mouse strains were spotted and immobilised onto amino-modified glass slides to generate a microarray. This was then interrogated through hybridisation with dual-colour probes, to determine the SNP genotypes of each sample. The results indicated that this microarray-based method could effectively determine the genotypes of the four selected SNPs with a high degree of accuracy. We have developed a new SNP genotyping technique for effective use in the genetic monitoring of inbred mouse strains.

A re-emerging strategy in the search for disease susceptibility genes is the evaluation of candidate genes, which are thought to play a role in disease pathogenesis. Candidate genes are screened for single nucleotide polymorphisms (SNPs) in a case-control study. The factor V Leiden (FVL) mutation (1691G --> A in the F5 gene) is an important risk factor for venous thrombosis. We asked ourselves whether the FVL mutation would have been found using the candidate gene approach in the absence of prior knowledge of the haplotype structure of the F5 gene. We typed four SNPs in the F5 gene in the Leiden Thrombophilia study, that is, promoter (99930G --> A), exon 13 (55907A --> G), exon 16 (42855A --> G), and intron 19 (37833T --> G). These SNPs were known to have different population frequencies, making their presence in distinct haplotypes likely. None of these SNPs has previously been associated with venous thrombotic risk. Subsequently we derived haplotypes. One haplotype was clearly more frequent in patients than controls (GAAT; 20 versus 9%), suggesting that a polymorphism in or near the F5 gene in this haplotype is associated with an increased thrombotic risk. If we had sequenced the F5 gene in patients homozygous for this haplotype, in order to locate the possible causal polymorphism, we would have found that 16 (76%) patients were homozygous or heterozygous for a missense mutation in exon 10 (1691G --> A), which predicts the replacement of Arg506 by Gln in one of the cleavage sites for activated protein C, a mutation that we now know as the FVL mutation.

Background: As life expectancy of HIV-infected patients increases with use of highly active antiretroviral therapy (HAART), protean haematologic manifestation including decreased activity of natural anticoagulants such as protein C may occur in the absence of genetic risk factors. Based on this preposition, we assessed the plasma level of protein C, and prevalence of factor V Leiden mutation among HIV-infected individuals. Our cohort consisted of 499 HIV-infected patients, of which 250 had AIDS, while 249 were either asymptomatic or had minor mucocutaneous infection consistent with WHO clinical stages I and II without features of AIDS. We also evaluated 251 healthy, HIV-negative subjects as controls. All participants were tested for plasma protein C levels and factor V Leiden (FVL) mutation (Arg 506 Gln) by automation and amplification created restriction enzyme site (ACRES) polymerase chain reaction, respectively. The prevalence of reduced protein C plasma levels among HIV positive patients was 20%; it was more prevalent among those that had AIDS compared with those without features of AIDS, but within WHO clinical stage I and II, (93.3% vs 6.7%) respectively. None of the control patients had either reduced protein C nor FVL mutation. All participants that demonstrated reduced protein C plasma levels demonstrated normal FVL genotype (1691G/G). Conclusion: Decreased protein C plasma levels can occur in HIV-infected patients in the absence of factor V Leiden mutation. The risk increases with severity of the disease. Deranged protein C plasma level increases the risk of hypercoagulable state in patients with advanced HIV disease; it should be considered among the causes of thrombo embolism in this group of patients.

Objectives: More evidence indicates that the G1691A mutation in the factor V Leiden (FVL) gene might be associated with susceptibility to breast cancer in humans being. Breast cancer is gradually becoming the most common cancer in Indian women, pathogenesis can be influenced by single nucleotide polymorphisms and several studies in the past have identified different genetic variants in the human genome that showed strong or moderate evidence of associations. FVL is a missense mutation, result of an amino acid substitution of glutamine for arginine at 506 position in the factor V molecule at nucleotide 1691 substitutes G for A, resulting in a mutant protein resistant to the anticoagulant action of activated protein C. Methods: We aimed to investigate the role of G1691A mutation in FVL gene and breast cancer women from south Indian population. One hundred cases and 100 controls were included in this study. DNA was separated and PCR-RFLP was performed followed by 2% electrophoresis. Results: The results of this study indicates that FVL mutation is associated with breast cancer patients (p<0.05). Conclusion: Our results concluded that FVL mutation has a role in breast cancer women and these results are supported by prior studies.

We presented here the application of two in-plate SNP (single nucleotide polymorphism) genotyping platforms for soybean plants [Glycine max (L.) Merr.], KASP® (Kompetitive Allele Specific PCR genotyping, LGC Genomics) and TaqMan® (Life Technologies) respectively. These two systems offer us an ability to determine the genotypes of 384 individual samples accurately and efficiently by allele specific PCR in a single plate using typical PCR conditions. Both of the systems require small quantity of genomic DNA obtained from a simple DNA extraction. The genomic sequences containing target SNPs can easily be used as a basic blueprint to design the probes and primers of KASP® and TaqMan® assays whether the sequences are obtained from the genome sequence of soybean William 82 (Wm82.a2.v1), Illumina Soy50k SNPs, or parallel resequencing. Moreover, we listed the pros and cons of the two systems and explained the principles behind the platforms. The high call rate and clear clustering separation of the SNPs can be readily obtained from these platforms without conducting any assay optimization processes. These platforms can routinely be performed on 96/384-well plate format with or without an automation procedure. Therefore, these platforms are especially suitable for the SNP genotyping on a particular trait with a large sample size, gene fine mapping, and marker assisted selection. Further, they require little hands-on experience and achieve per-site and per-individual costs below that of current SSR, AFLP, RFLP, and SNP chip technologies. The platforms can be used for genotyping on a wide range of organisms due to their simplicity and flexibility of handling. Meanwhile, we also especially presented some of the advantages using KASP® SNP genotyping pipeline, which was cost effective in the selection of allele specific assay and therefore, efficiently facilitated the soybean genotyping across large numbers (thousands or more) of individual lines for a great range of markers (hundreds to thousands) in our laboratory.

Genotyping of multiple single nucleotide polymorphisms with hyperbranched rolling circle amplification and microarray

Objectives: Several rheumatoid arthritis (RA) susceptibility loci have also been found to be associated with psoriatic arthritis (PsA), demonstrating that there is a degree of genetic overlap between various autoimmune diseases. We sought to investigate whether single nucleotide polymorphisms (SNPs) mapping to previously reported RA and/or PsA susceptibility loci, including PLCL2, CCL21, REL, STAT4, CD226, PTPN22, and TYK2, are associated with risk for the two diseases in a genetically homogeneous Greek population.Method: This study included 392 RA patients, 126 PsA patients, and 521 healthy age- and sex-matched controls from Greece. Genotyping of the SNPs was performed with Taqman primer/probe sets. Bioinformatic analysis was performed using BlastP, PyMOL, and Maestro and Desmond.Results: A significant association was detected between the GC genotype of rs34536443 (TYK2) in both the PsA and RA cohorts. The C allele of this SNP was associated with PsA only. Evidence for association with PsA was also found for the GG genotype and G allele of the rs10181656 SNP of STAT4. The TC genotype of the rs763361 SNP of CD226 was associated with PsA only.Conclusions: Genetic overlap between PsA and RA was detected for the rs34536443 SNP of the TYK2 gene within a Greek population. An association of STAT4 (rs10181656) with PsA was confirmed whereas CD226 (rs763361) was associated with PsA but not with RA, in contrast to previous reports. The different findings of this study compared to previous ones highlights the importance of comparative studies that include various ethnic or racial populations.

Simultaneous genotyping of multiplex single nucleotide polymorphisms of the K-ras gene with a home-made kit

A Case-Control Study of Candidate Immunoregulatory Genes Reveals Haplotypes That Influence Inhibitor Risk in Severe Hemophilia A.

Rapid one-pot human single nucleotide polymorphism genotyping platform with Cas13a nuclease

Among the most prevalent neurodevelopmental disorders, Autism Spectrum Disorder (ASD) is highly diverse showing a broad phenotypic spectrum. ASD also couples with a broad range of mutations, both de novo and inherited. In this study, we used a proprietary SNP genotyping chip to analyze the genomic DNA of 250 Vietnamese children diagnosed with ASD. Our Single Nucleotide Polymorphism (SNP) genotyping chip directly targets more than 800 thousand SNPs in the genome. Our primary focus was to identify pathogenic/likely pathogenic mutations that are potentially linked to more severe symptoms of autism. We identified and validated 23 pathogenic/likely pathogenic mutations in this initial study. The data shows that these mutations were detected in several cases spanning multiple biological pathways. Among the confirmed SNPs, mutations were identified in genes previously known to be strongly associated with ASD such as SLCO1B1, ACADSB, TCF4, HCP5, MOCOS, SRD5A2, MCCC2, DCC, and PRKN while several other mutations are known to associate with autistic traits or other neurodevelopmental disorders. Some mutations were found in multiple patients and some patients carried multiple pathogenic/likely pathogenic mutations. These findings contribute to the identification of potential targets for therapeutic solutions in what is considered a genetically heterogeneous neurodevelopmental disorder.

Noninvasive genetic studies of wild animals enable the recovery of information infeasible to obtain using other means. However, the low quantity and quality of noninvasively collected DNA often challenge the retrieval of reliable genotypes, which may cause biases in downstream analyses. In this study, we optimized SNP (single nucleotide polymorphism) genotyping of fecal samples from moose (Alces alces) with the main purpose of exploring the potential of using noninvasively retrieved genotypes for individual- and sex identification. Fecal pellets were collected during the late winter of 2016 on the Swedish island of Öland in the Baltic Sea and DNA was extracted and genotyped using 86 autosomal, six sex-specific and five species diagnostic SNPs. The SNP error rate of the quality filtered dataset was 0.06 and the probability of identity for siblings below 0.001. Following a thorough quality filtering process, 182 reliable genotypes were obtained, corresponding to 100 unique individuals (37 males, 63 females), with an estimated male proportion of 37% (± 9%). The population size, estimated using two different capture-mark-recapture approaches, was found to be in the range of 115–156 individuals (95% CI). Furthermore, moose on Öland showed significantly lower heterozygosity levels (zHexp = −5.51, N = 69, pHexp = 3.56·10−8, zHobs = −3.58, N = 69, pHobs = 3.38·10−4) and appeared genetically differentiated from moose on the Swedish mainland. Thus, we show that quality controlled noninvasively derived SNP genotypes can be highly informative for individual and population monitoring in a large ungulate.

Objective : The reports about Factor V (FV) Leiden, Factor II (FII) G20210A and Methylenetetrahydrofolate reductase (MTHFR) C677T gene mutations of parents and fetal viability are frequently encountered in the literature, despite the fetal side of thrombophilia is scant. To clarify the three common thrombophilic gene mutations of the spontaneously aborted embryos, an accurate algorithm was followed to extract the fetal tissues and then the mutations were searched. Material and Methods : 70 spontaneous abortion materials were included to the study and all were karyotyped. Cytogenetically abnormals were excluded from the study. To extract the fetal tissues, amplifications of sex determination region of chromosome-Y (SRY) gene and genotypings were performed, respectively. Extracted fetal tissues of spontaneous aborted embroys and parents were screened for the thrombophilic gene mutations via electronic microarray. Results: After excluding chromosomally abnormal and maternally contaminated ones totally ten fetal tissues were screened for the FII G20210A, FV Leiden and MTHFR C677T gene mutations, and two carry F II G20210A and F Leiden heterozygote mutations, and six carry heterozygote forms of MTHFR C677T. Conclusion : The present study performed on the limited number of abortion materials, has a value for distinguishing the fetal tissues before analyzing the three common mutation of thrombophilic genes which make the results are very substantial.

An electrochemical aptasensor for exosomes based on strand displacement amplification and hybridization chain reaction amplification