PrimerStation: a highly specific multiplex genomic PCR primer design server for the human genome

PrimerStation () is a web service that calculates primer sets guaranteeing high specificity against the entire human genome. To achieve high accuracy, we used the hybridization ratio of primers in liquid solution. Calculating the status of sequence hybridization in terms of the stringent hybridization ratio is computationally costly, and no web service checks the entire human genome and returns a highly specific primer set calculated using a precise physicochemical model. To shorten the response time, we precomputed candidates for specific primers using a massively parallel computer with 100 CPUs (SunFire 15 K) about 3 months in advance. This enables PrimerStation to search and output qualified primers interactively. PrimerStation can select highly specific primers suitable for multiplex PCR by seeking a wider temperature range that minimizes the possibility of cross-reaction. It also allows users to add heuristic rules to the primer design, e.g. the exclusion of single nucleotide polymorphisms (SNPs) in primers, the avoidance of poly(A) and CA-repeats in the PCR products, and the elimination of defective primers using the secondary structure prediction. We performed several tests to verify the PCR amplification of randomly selected primers for ChrX, and we confirmed that the primers amplify specific PCR products perfectly

Automatic Volumetry of the Cerebrospinal Fluid Space in Idiopathic Normal Pressure Hydrocephalus

Objectives: To measure the cerebrospinal fluid (CSF) space volume in idiopathic normal pressure hydrocephalus (INPH), we developed a software that allows us to automatically measure the regional CSF space and compared the volumes of the ventricle systems (VS), Sylvian fissures (SF) and sulci at high convexity and midline (SHM) among INPH patients, Alzheimer's disease (AD) patients and healthy volunteers (HVs). Methods: Fifteen INPH patients, 15 AD patients and 15 HVs were retrospectively selected for this study. 3D-T1 MR images were obtained. We improved upon an automatic gray matter volume system to measure CSF spaces, adopting new regions for the template of INPH-characteristic CSF spaces and measured them. The VS, SF and SHM volumes were calculated relative to the intracranial volume. Results: The relative SHM volume of the INPH group (0.0237 ± 0.0064) was the smallest among the 3 groups (AD: 0.0477 ± 0.0109, HV: 0.0542 ± 0.0045). The VS (0.0499 ± 0.0135) and SF (0.0187 ± 0.0037) volumes of the INPH group were significantly larger than those of the AD (VS: 0.0311 ± 0.0075, SF: 0.0146 ± 0.0026) and HV groups (VS: 0.0167 ± 0.0065, SF: 0.0111 ± 0.017). Conclusion: Automatic volume measurement can be used to delineate the characteristic changes in CSF space in patients with INPH and is useful in the diagnosis of INPH

Altered Quality Control in the Endoplasmic Reticulum Causes Cortical Dysplasia in Knock-In Mice Expressing a Mutant BiP▿

Binding immunoglobulin protein (BiP) is an endoplasmic reticulum (ER) molecular chaperone that is central to ER function. We examined knock-in mice expressing a mutant BiP in order to elucidate physiological processes that are sensitive to BiP functions during development and adulthood. The mutant BiP lacked the retrieval sequence that normally functions to return BiP to the ER from the secretory pathway. This allowed us to examine the effects of a defect in ER function without completely eliminating BiP function. The homozygous mutant BiP neonates died after birth due to respiratory failure. Besides that, the mutant BiP mice displayed disordered layer formation in the cerebral cortex and cerebellum, a neurological phenotype of reeler mutant-like malformation. Consistent with the phenotype, Cajal-Retzius (CR) cells did not secrete reelin, and the expression of reelin was markedly reduced posttranscriptionally. Furthermore, the reduction in the size of the whole brain and the apparent scattering of CR cells throughout the cortex, which were distinct from the reeler phenotype, were also seen. These findings suggest that the maturation and secretion of reelin in CR cells and other factors related to neural migration may be sensitive to aberrant ER quality control, which may cause various neurological disorders

Rapid detection of SNP (c.309T>G) in the MDM2 gene by the Duplex SmartAmp method.

BACKGROUND: Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP) c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans. METHODOLOGY: In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named "Duplex SmartAmp," which enabled us to simultaneously detect both 309T and 309G alleles in one tube. To develop this new method, we introduced new primers i.e., nBP and oBPs, as well as two different fluorescent dyes that separately detect those genetic polymorphisms. RESULTS AND CONCLUSIONS: By the Duplex SmartAmp method, the genetic polymorphisms of the MDM2 gene were detected directly from a small amount of genomic DNA or blood samples. We used 96 genomic DNA and 24 blood samples to validate the Duplex SmartAmp by comparison with results of the conventional PCR-RFLP method; consequently, the Duplex SmartAmp results agreed totally with those of the PCR-RFLP method. Thus, the new SNP detection method is considered useful for detecting the SNP c.309T>G in the MDM2 gene so as to judge cancer susceptibility against some cellular stress in the clinical setting, and also to handle a large number of samples and enable rapid clinical diagnosis