Cell-free DNA Oncogene Copy Number as a Surrogate Molecular Biomarker in ALK/MYCN-coamplified Neuroblastoma.

Secondary expansion and/or evolution of aggressive subclones are associated with the disease progression and resistance to chemotherapy in neuroblastoma, and it is important to track the clonal changes during the treatment period. Cell-free (cf) DNA analysis, namely liquid biopsy, can detect the genomic change of tumor cells without surgical procedures. In this report, we showed that serial polymerase chain reaction-based cf DNA neuroblastoma proto-oncogene quantification is sensitive enough to evaluate the aggressive cellular characteristics of ALK/MYCN-coamplified neuroblastoma and stressed the promise of cf DNA analyses as a reliable molecular marker in advanced neuroblastoma.

EFFECTS OF MOUTHING AND INTERLOCUTOR PRESENCE ON MOVEMENTS OF VISIBLE VS. NON-VISIBLE ARTICULATORS.

Speakers take into account what information a conversation partner requires in a given context in order to best understand an utterance. Despite growing evidence showing that movements of visible articulators such as the lips are augmented in mouthed speech relative to vocalized speech, little to date has been done comparing this effect in visible vs. non-visible articulators. In addition, no studies have examined whether interlocutor engagement differentially impacts these. Building on a basic present/not-present design, we investigated whether presence of audible speech information and/or an interlocutor affect the movements of the lips and the tongue. Participants were asked to a) speak or b) mouth three target syllables in interlocutor-present and interlocutor-not-present conditions, while lip and tongue movements were recorded using video and ultrasound imaging. Results show that lip protrusion was greater in mouthed conditions compared to vocalized ones and tongue movements were either attenuated (/wa/) or unaffected (/ri/, /ra/) by these same conditions, indicating differential effects for the visible and non-visible articulators in the absence of an auditory signal. A significant interaction between the social engagement and vocalizing conditions in reference to lip aperture showed that participants produced smaller lip apertures when vocalizing alone, as compared to when in the presence of an interlocutor. However, measures of lip protrusion failed to find an effect of social engagement. We conclude that speakers make use of both auditory and visual modalities in the presence of an interlocutor, and that when acoustic information is unavailable, compensatory increases are made in the visual domain. Our findings shed new light on the multimodal nature of speech, and pose new questions about differential adaptations made by visible and non-visible articulators in different speech conditions.

Les locuteurs prennent en compte l'information qu'un partenaire de conversation nécessite pour mieux comprendre une expression. Malgré l'évidence grandissante que les mouvements d'articulateurs visibles (comme les lèvres) sont augmentés dans l'articulation silencieuse par rapport à l'articulation vocalisée, peux d'études ont comparé cet effet dans les articulateurs visibles contre les articulateurs non visibles. De plus, aucune étude n'a examiné si l'engagement de l'interlocuteur changera ces résultats. En élaborant un conception d'expérience présent/non présent, nous avons testé si la présence d'information audible et/ou d'un interlocuteur affecte les mouvements des lèvres et de la langue. Les participants ont parlé trois syllabes, avec et sans production audible, dans chacune des conditions interlocuteur-présent et interlocuteur-non présent. Les mouvements des lèvres et de la langue étaient enregistrés avec la vidéo et l'échographie. Nos résultats montrent que la protubérance des lèvres était plus grande dans les conditions non audibles par rapport à ceux audibles et que les mouvements de la langue étaient atténués (/wa/) ou non affectés (/ri/, /ra/) par ces mêmes conditions, indiquant les effets différents pour les articulateurs visibles et non-visibles dans l'absence d'un signal auditif. Une interaction significative entre les conditions d'engagement sociale et d'audibilité de vocalisation avec référence à la fermeture orale a montré que les participants ont produit des fermetures plus étroites dans les conditions de vocalisation audible, interlocuteur-non présent (par rapport à la condition interlocuteur-présent). Cependant, les mesures de protubérance des lèvres n'étaient pas affectées par condition d'engagement sociale. Nous concluons que les locuteurs utilisent à la fois les modalités auditives et visuelles dans la présence d'un interlocuteur, et lorsque l'information acoustique n'est pas disponible, les augmentations compensatoires sont réalisés dans le domain visuel. Nos résultats soulignent encore le caractère multimodal de discours, et posent des nouvelles questions au sujet des adaptations différentielles faites par les articulateurs visibles et non visibles dans les différentes conditions de parole.

Off-camera gaze decreases evaluation scores in a simulated online job interview.

During the pandemic, digital communication became paramount. Due to the discrepancy between the placement of the camera and the screen in typical smartphones, tablets and laptops, mutual eye contact cannot be made in standard video communication. Although the positive effect of eye contact in traditional communication has been well-documented, its role in virtual contexts remains less explored. In this study, we conducted experiments to gauge the impact of gaze direction during a simulated online job interview. Twelve university students were recruited as interviewees. The interview consisted of two recording sessions where they delivered the same prepared speech: in the first session, they faced the camera, and in the second, they directed their gaze towards the screen. Based on the recorded videos, we created three stimuli: one where the interviewee's gaze was directed at the camera (CAM), one where the interviewee's gaze was skewed downward (SKW), and a voice-only stimulus without camera recordings (VO). Thirty-eight full-time workers participated in the study and evaluated the stimuli. The results revealed that the SKW condition garnered significantly less favorable evaluations than the CAM condition and the VO condition. Moreover, a secondary analysis indicated a potential gender bias in evaluations: the female evaluators evaluated the interviewees of SKW condition more harshly than the male evaluators did, and the difference in some evaluation criteria between the CAM and SKW conditions was larger for the female interviewees than for the male interviewees. Our findings emphasize the significance of gaze direction and potential gender biases in online interactions.

Cloning and sequencing analysis of whole Spiroplasma genome in yeast.

Cloning and transfer of long-stranded DNA in the size of a bacterial whole genome has become possible by recent advancements in synthetic biology. For the whole genome cloning and whole genome transplantation, bacteria with small genomes have been mainly used, such as mycoplasmas and related species. The key benefits of whole genome cloning include the effective maintenance and preservation of an organism's complete genome within a yeast host, the capability to modify these genome sequences through yeast-based genetic engineering systems, and the subsequent use of these cloned genomes for further experiments. This approach provides a versatile platform for in-depth genomic studies and applications in synthetic biology. Here, we cloned an entire genome of an insect-associated bacterium, Spiroplasma chrysopicola, in yeast. The 1.12 Mbp whole genome was successfully cloned in yeast, and sequences of several clones were confirmed by Illumina sequencing. The cloning efficiency was high, and the clones contained only a few mutations, averaging 1.2 nucleotides per clone with a mutation rate of 4 × 10-6. The cloned genomes could be distributed and used for further research. This study serves as an initial step in the synthetic biology approach to Spiroplasma.

VS38 staining contributes to a novel gating strategy in flow cytometry for small B cell lymphoma, especially in lymphoplasmacytic lymphoma/Waldenström macroglobulinemia.

BACKGROUND: Multi-parametric flow cytometry (MFC) is a helpful tool for detecting neoplastic cells in malignant lymphoma; however, lymphoma cells can be difficult to detect when characteristic immunophenotypic abnormalities are not evident. We evaluated the stainability of VS38, which is used for multiple myeloma, in normal and abnormal B cells using MFC to develop a new strategy for detecting lymphoma cells. METHODS: We compared the median fluorescence intensity of VS38 staining in lymphocytes from patients without hematopoietic neoplasms and in B cells from 26 patients with B cell lymphoma (BCL). To evaluate the performance of VS38 gating, we compared VS38-positive B cells with the percentages of BCL cells, and with the mutation ratios of MYD88 L265P measured by droplet digital PCR in patients with lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM). RESULTS: CD27-positive memory B cells were stained with VS38, whereas normal lymphocytes were faintly stained. Lymphoma cells were stained with VS38 in 11 of 12 patients with LPL/WM, 3 of 3 with chronic lymphocytic leukemia, 3 of 5 with mantle cell lymphoma, 2 of 4 with follicular lymphoma, and 1 of 1 with splenic marginal zone lymphoma. The percentages of VS38-positive B cells in VS38-positive BCL were equivalent to those of lymphoma cells and the mutation ratios of MYD88 L265P in LPL/WM. CONCLUSIONS: VS38 identified neoplastic cells in plasma cell disorders and BCL. This might improve the accuracy of BCL diagnosis, especially in patients with LPL/WM.

Sensitive detection of GATA1 mutations using complementary DNA-based analysis for transient abnormal myelopoiesis associated with the Down syndrome.

INTRODUCTION: GATA1 mutation plays an important role in initiating transient abnormal myelopoiesis (TAM) and in the clonal evolution towards acute megakaryoblastic leukaemia (AMKL) associated with Down syndrome (DS). This study aimed to develop and validate the clinical utility of a complementary DNA (cDNA) analysis in parallel with the conventional genomic DNA (gDNA) Sanger sequencing (Ss), as an initial screening test for GATA1 mutations. METHODS: GATA1 mutations were evaluated using both gDNA and cDNA in 14 DS patients using Ss and fragment analysis (FA), respectively. RESULTS: The detection sensitivity of conventional gDNA sequencing was limited in low blast percentage TAM (LBP-TAM); however, cDNA-based Ss readily detected all the pathognomonic GATA1 mutations. The cDNA-based FA readily detected GATA1 frameshift mutation with a reliable sensitivity ranging from 0.005% to 0.01% of clonal cells. CONCLUSIONS: GATA1 mutations are heterogeneous; therefore, we would like to propose a dual cDNA and gDNA analysis as a standard diagnostic approach, especially for LBP-TAM. cDNA-based FA promises an excellent sensitivity for detecting frameshift GATA1 mutations in the longitudinal clonal evolution towards AMKL without using a patient specific primer.

Reciprocal association of serum Mac-2 binding protein and HDL-cholesterol concentrations.

BACKGROUND: Mac-2 binding protein (Mac-2BP) is used as a serum biomarker of nonalcoholic steatohepatitis, considered to be a liver phenotype of metabolic syndrome (MetS). In this study, we investigated the serum Mac-2BP concentrations-correlated MetS-related clinical parameters in vivo, and the underlying mechanism in vitro. MATERIALS & METHODS: We enrolled 54 healthy Japanese men who underwent health examination at Osaka University Health Care Center in this study. Physical and serum biochemical parameters were obtained from all the subjects. In the cultured HepG2 cells, the effects of interferon (IFN)-γ on the expression of Mac-2BP, apolipoprotein (apo) A-I, and ATP binding cassette transporter A1 (ABCA1) were studied. RESULTS: Serum Mac-2BP concentrations correlated negatively with HDL-C, and positively with body mass index and systolic blood pressure in univariate analysis. These results suggested the association between Mac-2BP and MetS, although none of these 3 parameters had significant correlation with serum Mac-2BP concentrations in multivariate analysis. In HepG2 cells, IFN-γ stimulation resulted in the increased Mac-2BP and the decreased ABCA1 and apo A-I mRNA concentrations, while Mac-2BP had no effects on ABCA1 and apo A-I concentrations. CONCLUSIONS: The serum Mac-2BP concentrations are negatively correlated with HDL-C concentrations in healthy subjects, as a result of chronic inflammation.

Identification and characterization of genes responsible for biosynthesis of kojic acid, an industrially important compound from Aspergillus oryzae.

Kojic acid is produced in large amounts by Aspergillus oryzae as a secondary metabolite and is widely used in the cosmetic industry. Glucose can be converted to kojic acid, perhaps by only a few steps, but no genes for the conversion have thus far been revealed. Using a DNA microarray, gene expression profiles under three pairs of conditions significantly affecting kojic acid production were compared. All genes were ranked using an index parameter reflecting both high amounts of transcription and a high induction ratio under producing conditions. After disruption of nine candidate genes selected from the top of the list, two genes of unknown function were found to be responsible for kojic acid biosynthesis, one having an oxidoreductase motif and the other a transporter motif. These two genes are closely associated in the genome, showing typical characteristics of genes involved in secondary metabolism.

Sibilant Fricative Merging in Taiwan Mandarin: An Investigation of Tongue Postures using Ultrasound Imaging.

In Taiwan Mandarin, retroflex [ʂ] is allegedly merging with dental [s], reducing the traditional three-way contrast between sibilant fricatives (i.e., dental [s]-retroflex [ʂ]-alveopalatal [ɕ]) to a two-way contrast. Most of the literature on the observed merging focuses on the acoustic properties and perceptual identification of the sibilants, whereas much less attention has been drawn to the articulatory evidence accounting for the aforementioned sibilant merging. The current study employed ultrasound imaging techniques to uncover the tongue postures for the three sibilant fricatives [s, ʂ, ɕ] in Taiwan Mandarin occurring before vowels [a], [ɨ], and [o]. Results revealed varying classes of the [s-ʂ] merger: complete merging (overlap), no merging (non-overlap), and context-dependent merging (context-dependent overlap, which only occurred before [a]). The observed [s-ʂ] merger was also confirmed by the perceptual identification by trained phoneticians. Center of gravity (CoG), a reliable spectral moment of identifying different sibilant fricatives, was also measured to reflect the articulatory-acoustic correspondence. Results showed that the [s-ʂ] merger varies across speakers and may also be conditioned by vowel contexts and that articulatory mergers may not be entirely reflected in CoG values, suggesting that auxiliary articulatory gestures may be employed to maintain the acoustic contrast.

cDNA-Based Mutation Screening Using a Combination of High-Resolution Melting Curve and Fragment Analysis Facilitates Efficient CCR4 Mutation Analysis in Adult T-Cell Leukemia/Lymphoma.

OBJECTIVES: C-C chemokine receptor type 4 (CCR4) proteins are expressed on the neoplastic cells of adult T-cell leukemia/lymphoma (ATLL). As the mutation status of CCR4 gene is reported to correlate with significant clinical information such as prognosis and response to mogamulizumab, we aimed to establish a screening method that is suitable for clinical laboratory tests. METHODS: In 34 patients with ATLL, CCR4 mutation analysis, high-resolution melting (HRM) analysis, fragment analysis, and direct sequencing were performed using both genomic DNA and complementary DNA (cDNA). Furthermore, 38 cases of asymptomatic carriers of human T-cell leukemia virus type 1 (HTLV-1) were screened for CCR4 mutation. RESULTS: Mutation analysis by direct sequencing of 34 ATLL clinical samples detected CCR4 mutation in four genomic DNA samples and seven cDNA samples, and two novel mutations were identified. All CCR4 mutations detected by direct sequencing were positive for HRM analysis and/or fragment analysis. CCR4 mutation was not detected in the asymptomatic carriers of HTLV-1. CONCLUSIONS: CCR4 mutation screening by a combination of HRM and fragment analysis using cDNA is a simple and practical method, and it will contribute to better decision making for a therapeutic strategy, providing a rapid CCR4 mutational status to clinicians.

Evaluation of target specificity of antibacterial agents using Staphylococcus aureus ddlA mutants and D-cycloserine in a silkworm infection model.

The availability of a silkworm larva infection model to evaluate the therapeutic effectiveness of antibiotics was examined. The 50% effective doses (ED50) of D-cycloserine against the Staphylococcus aureus ddlA mutant-mediated killing of larvae were remarkably lower than those against the parental strain-mediated killing of larvae. Changes in MICs and ED50 of other antibiotics were negligible, suggesting that these alterations are d-cycloserine selective. Therefore, this model is useful for selecting desired compounds based on their therapeutic effectiveness during antibiotic development.

Evaluation of SF3B1 Mutation Screening by High-Resolution Melting Analysis and its Clinical Utility for Myelodysplastic Syndrome with Ring Sideroblasts at the Point of Diagnosis.

BACKGROUND: SF3B1 (splicing factor 3B subunit-1) somatic mutation is specifically detected in myelodysplastic syndrome (MDS) with ring sideroblasts (MDS-RS). We investigated the sensitivity and utility of SF3B1 mutation analysis as a clinical laboratory test. METHOD: Detection limit for SF3B1 mutations by high-resolution melting (HRM) analysis was investigated by plasmid mixture. In 67 MDS patients, we examined the association between SF3B1 mutation and prognostic evaluation using the Revised International Prognostic Scoring System and revalidated MDS classifications based on the revised 4th edition of the WHO classification. RESULTS: HRM analysis enabled mutation detection in the 12.5% SF3B1 mutant alleles. SF3B1 mutation was detected in 9 cases, mostly in the low-risk group. Cases of MDS with ring sideroblasts unrelated to SF3B1 mutation were detected in the high-risk group. Two cases were reclassified as MDS-RS after detecting SF3B1 mutation. CONCLUSIONS: SF3B1 mutation analysis as an initial screening at diagnosis increases the accuracy of prognostic prediction and disease classification.

VS38 as a promising CD38 substitute antibody for flow cytometric detection of plasma cells in the daratumumab era.

The development of effective therapies has enabled long-term survival for many patients with multiple myeloma (MM). However, the administration of antibody drugs, such as daratumumab, which bind to plasma cell (PC) surface proteins, may prevent PC detection by flow cytometry. We propose VS38 as an alternative antibody for CD38. VS38 recognizes cytoskeleton-linking membrane protein 63 (CLIMP-63) on the rough endoplasmic reticulum, and this protein may be expressed in secretory cells. We investigated VS38 staining in normal hematopoietic cells from five control samples, as well as PCs from 21 patients with plasma cell disorder (PCD). In normal hematopoietic cells, although VS38-stained monocytes, myeloid cells, and a subpopulation of B cells, PCs were significantly and brightly stained by VS38. There was no significant difference in VS38 staining between normal and abnormal PCs obtained from five patients with monoclonal gammopathy of undetermined significance. Furthermore, PCs in 21 PCD cases were clearly identified by VS38 in all cases, in contrast to CD38, even in daratumumab-administered patients whose CD38 epitopes on PCs were masked. These results suggest that the use of the VS38 antibody in flow cytometry contributes to PC detection, independent of therapeutic treatment.

Investigation of screening method for DNMT3A mutations by high-resolution melting analysis in acute myeloid leukemia.

INTRODUCTION: Acute myeloid leukemia (AML) is a heterogeneous disease associated with various genetic abnormalities. Somatic mutations in nucleophosmin 1 (NPM1), fms-related tyrosine kinase 3 (FLT3), and DNA methyltransferase 3 alpha (DNMT3A) are the most frequent mutations associated with AML. However, because DNMT3A mutations are broadly distributed, they are challenging to analyze in routine laboratory tests. Hence, we developed a rapid screening method for DNMT3A mutations by high-resolution melting (HRM) analysis for clinical use at the point of AML diagnosis. METHODS: The detection limit for DNMT3A mutations from exons 8-23 by an HRM analysis was investigated using plasmid mixtures. In 69 patients with AML, somatic mutations in NPM1, FLT3-internal tandem duplication (ITD), FLT3-tyrosine kinase domain (TKD), DNMT3A, and isocitrate dehydrogenase 1/2 were screened using HRM analysis, and direct sequencing was performed for positive samples. RESULTS: High-resolution melting analysis enabled complete mutation detection in samples with 20% mutant alleles in all regions. In a clinical laboratory test, DNMT3A mutations were detected in 12 cases (17.3%), and we identified five novel mutations. Simultaneous NPM1, FLT3-ITD, and DNMT3A mutations constituted the most common pattern (30%) in de novo cytogenetically normal AML. CONCLUSION: High-resolution melting analysis has sufficient performance for the detection of DNMT3A mutations in AML. This approach can facilitate rapid AML genotyping at diagnosis in clinical settings.

Transcriptional regulation of genes on the non-syntenic blocks of Aspergillus oryzae and its functional relationship to solid-state cultivation.

Transcriptome analysis revealed close relationship between solid-state cultivation and the transcriptional regulation of the genes on the non-syntenic blocks (NSBs), which were characterized by the comparison of Aspergillus oryzae genome with those of Aspergillus fumigatus and Aspergillus nidulans. Average expression ratio of the genes on NSBs in solid-state cultivation was significantly higher than that on the syntenic blocks (SBs). Of the induced genes, the genes relating to metabolism, which are highly enriched on NSBs, most contributed to the NSB-specific induction. The analysis using the SB- and NSB-genes that had sequence similarity between the two blocks significantly decreased the difference of average expression ratios between the two blocks. In spite of remarkably high averaged expression ratio of the NSB genes encoding extracellular enzymes, no induction of PKS and NRPS genes on NSBs were observed in solid-state cultivations. These results strongly suggest that the genes on NSBs play an important role on solid-state fermentation.

The effect of a novel, small non-peptidyl molecule butyzamide on human thrombopoietin receptor and megakaryopoiesis.

BACKGROUND: Thrombocytopenia is a common problem in the management of patients with cancer and other conditions that affect hematopoietic cells. In previous clinical trials, the polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor increased platelet counts in patients with idiopathic thrombocytopenic purpura and solid tumors undergoing chemotherapy. However, antibodies to polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor develop in healthy volunteers and patients undergoing chemotherapy and cross-react with endogenous thrombopoietin. As a result, clinical development of polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor was discontinued in 1998. The aim of this study was to identify an orally bioavailable human Mpl activator that does not develop autoantibodies against endogenous thrombopoietin. DESIGN AND METHODS: We screened our chemical library and created a novel non-peptidyl thrombopoietin receptor, Mpl activator named butyzamide. We evaluated the effect of butyzamide on megakaryopoiesis in vitro using Ba/F3 cells expressing Mpl and human hematopoietic stem cells. For the evaluation of its in vivo effect, we administered butyzamide orally to immunodeficient NOD/Shi-scid,IL-2R gamma(null) (NOG) mice transplanted with human fetal liver-derived CD34(+) cells and investigated the production of human platelets. RESULTS: Butyzamide specifically reacted with human Mpl and activated the same signal transduction pathway as thrombopoietin. However, unlike thrombopoietin, butyzamide did not react with murine Mpl and was shown to require the histidine residue in the transmembrane domain of Mpl for its agonistic activity. Butyzamide induced colony-forming unit-megakaryocytes and polyploid megakaryocytes from human CD34(+) hematopoietic progenitor cells, and its effects were comparable to those of thrombopoietin. When butyzamide was administered orally at the doses of 10 and 50 mg/kg for 20 days to NOG mice transplanted with human fetal liver-derived CD34(+) cells, the human platelet count increased by 6.2- and 22.9-fold, respectively. CONCLUSIONS: Butyzamide is an orally bioavailable human Mpl activator, and appears to have potential for clinical development as a therapeutic agent for patients with thrombocytopenia.

Characterization of novel non-peptide thrombopoietin mimetics, their species specificity and the activation mechanism of the thrombopoietin receptor.

A series of non-peptide small compounds discovered to be thrombopoietin receptor agonists showed species specificity to humans. Compound I could induce megakaryocyte lineage from human bone marrow cells, but not from mouse, guinea pig or cynomolgus monkey bone marrow cells. To elucidate the mechanism, we identified the pivotal amino acid residue for the receptor activation by compound I by taking advantage of its species specificity. The response of compound I to three human/mouse chimeric receptors indicated the importance of the transmembrane domain. Comparison of amino acid sequences of the transmembrane domain of the thrombopoietin receptor between human and three animal species led us to hypothesize that histidine 499 is necessary for the reactivity to the thrombopoietin mimetics. We verified the hypothesis using two mutant receptors: the human thrombopoietin receptor mutant His499Leu and the mouse thrombopoietin receptor mutant Leu490His. These results should be helpful for structure-activity relationship studies and conducting in vivo studies of thrombopoietin mimetics.

Discovery of novel non-peptide thrombopoietin mimetic compounds that induce megakaryocytopoiesis.

We have identified a series of novel non-peptide compounds that activate the thrombopoietin-dependent cell line Ba/F3-huMPL. The compounds stimulated proliferation of Ba/F3-huMPL in the absence of other growth factors, but did not promote proliferation of the thrombopoietin-independent parent cell line Ba/F3. The thrombopoietin-mimetic compounds elicited signal-transduction responses comparable with recombinant human thrombopoietin, such as tyrosine phosphorylation of the thrombopoietin receptor, JAK (Janus kinase) 2, Tyk2 (tyrosine kinase 2), STAT (signal transducer and activator of transcription) 3, STAT5, MAPKs (mitogen-activated protein kinases), PLCgamma (phospholipase Cgamma), Grb2 (growth-factor-receptor-bound protein 2), Shc (Src homology and collagen homology), Vav, Cbl and SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) and increased the number of CD41(+) cells (megakaryocyte lineage) in cultures of human CD34(+) bone-marrow cells (haematopoietic stem cells). These findings suggest that this series of compounds are novel agonists of the human thrombopoietin receptor and are possible lead compounds for the generation of anti-thrombocytopaenia drugs.

Development of an automated SNP analysis method using a paramagnetic beads handling robot.

Biological and medical importance of the single nucleotide polymorphism (SNP) has led to development of a wide variety of methods for SNP typing. Aiming for establishing highly reliable and fully automated SNP typing, we have developed the adapter ligation method in combination with the paramagnetic beads handling technology, Magtration(R). The method utilizes sequence specific ligation between the fluorescently labeled adapter and the sample DNAs at the cohesive end produced by a type IIS restriction enzyme. Evaluation of the method using human genomic DNA showed clear discrimination of the three genotypes without ambiguity using the same reaction condition for any SNPs examined. The operations following PCR amplification were automatically performed by the Magtration(R)-based robot that we have previously developed. Multiplex typing of two SNPs in a single reaction by using four fluorescent dyes was successfully preformed at the almost same sensitivity and reliability as the single typing. These results demonstrate that the automated paramagnetic beads handling technology, Magtration(R), is highly adaptable to the automated SNP analysis and that our method best fits to an automated in-house SNP typing for laboratory and medical uses.