Unique Genotyping Protocol of CYP2D6 Allele Frequency Using Real Time Quantitative PCR from Japanese Healthy Women.

CYP2D6 is an important drug-metabolizing enzyme involved in the metabolism of 20-25% of commonly prescribed drugs. Genetic polymorphism of CYP has clinically significant modifications in patients' drug-metabolizing capacities. Since gene copy number variation (CNV) and single nucleotide polymorphism (SNP) frequently occur in the CYP2D6 gene, which the activity of CYP2D6 particularly depend on the genetic factors.

Development of an Inexpensive and Rapid Operation Device for High-Throughput Real-Time Quantitative PCR-Based CYP2D6 CNV Genotyping.

The CYP2D6 gene is the most well characterized gene involved in drug metabolism and is known to have both gene duplication and deletion variants. We report an optimized method for the determination of copy number variation (CNV) in the CYP2D6 gene by a novel purification process for a real-time quantitative PCR. This high-throughput low-cost method accurately determines CNV in the CYP2D6 gene enabling reliable estimates of disease prediction in large epidemiological samples.

Rapid and Cost-Effective Genotyping Protocol for Angiotensin-Converting Enzyme Insertion/Deletion (Ins/Del) Polymorphism from Saliva.

DNA extraction and purification have been generally considered to be required for PCR assay. We demonstrated a new protocol using biological specimens directly as templates for real-time PCR with melting curve analysis. We confirmed the melting curve analysis was particularly suitable for the identification of the insertion/deletion (Ins/Del) polymorphism of the angiotensin-converting enzyme (ACE) gene.

[Development of Novel Genotyping Protocol and Its Application for Genotyping of Alcohol Metabolism-related Genes].

A new single nucleotide polymorphisms (SNP) genotyping method has been developed and validated using biological specimens directly as templates for TaqMan PCR without general DNA extraction and purification procedure from dried saliva samples attached on water-soluble papers. This new method can set up at ease and complete PCR analysis including data interpretation in under two hours with additional advantages of application for large-scale clinical research, diagnostics, and epidemiological studies at low cost. Specifically, SNP genotyping of alcohol metabolism-related genes ADH1B (rs1229984) and ALDH2 (rs671) were demonstrated by TaqMan PCR assay using dried saliva samples in the present investigation.

Effects of ADH1B and ALDH2 Genetic Polymorphisms on Alcohol Elimination Rates and Salivary Acetaldehyde Levels in Intoxicated Japanese Alcoholic Men.

Background: The genetic polymorphisms of alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) are associated with the risk of alcoholism and upper aerodigestive tract cancer in alcoholics. Salivary ethanol (sEtOH) levels are well correlated with blood EtOH levels.

Methods: To study the effects of ADH1B and ALDH2 genotypes on the alcohol elimination rate (AER) and salivary acetaldehyde (sAcH) levels, we measured the sEtOH and sAcH levels twice at a 1-hour intervals in 99 intoxicated Japanese alcoholic men who had stopped drinking for 4 or more hours.

[Verification and Validation on Single Nucleotide Polymorphism Analysis of Alcohol Metabolism-Related Genes ADH1B and ALDH2, Using Dried-Saliva Samples].

We have developed a new method for unprocessed biological specimens as templates directly into the TaqMan assay. Saliva was needed to be put on a water-soluble paper and dried, because foreign substances, such as a filter paper, hinder fluorescence detection through the assay. Genotyping of alcohol metabolism-related genes ADH1B (rs1229984) and ALDH2 (rs671) polymorphisms was, subsequently, performed by TaqMan PCR assay using dried saliva in the present investigation.

[Associations between ALDH2 and ADH1B Genotypes and Ethanol-Induced Cutaneous Erythema in Young Japanese Women].

Objectives: The purpose of this study was to identify associations between ALDH2 and ADH1B genotypes and ethanol-induced cutaneous erythema and assess the accuracy of an ethanol patch test in young Japanese women.

Methods: The subjects were 942 female Japanese university students. They were given an ethanol patch test and examined for ethanol-induced cutaneous erythema both immediately after removing the patch and 10 minutes after removing the patch.

Combination analysis in genetic polymorphisms of drug-metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese population.

The Cytochrome P450 is the major enzyme involved in drug metabolism. CYP enzymes are responsible for the metabolism of most clinically used drugs. Individual variability in CYP activity is one important factor that contributes to drug therapy failure.

Direct detection of single nucleotide polymorphism (SNP) by the TaqMan PCR assay using dried saliva on water-soluble paper and hair-roots, without DNA extraction.

We have developed a new method for directly using unprocessed biological specimens as templates for the TaqMan assay. DNA extraction and purification had been believed to be required for the assay, but our new method could avoid hindering fluorescence detection, even if the templates were used directly. Saliva was needed to be put on water-soluble paper and dried, and hairs were cut to be about 10 mm long.

Long PCR-based genotyping for a deleted CYP2D6 gene without DNA extraction.

In the post-genome era, a simple and inexpensive method for diagnostic analysis is in high demand. Cytochrome P450 (CYP) 2D6 is one of the most widely investigated CYPs in relation to genetic polymorphism. Detection of CYP2D6*5 is difficult since long PCR is used.

Genotyping of polymorphisms in alcohol and aldehyde dehydrogenase genes by direct application of PCR-RFLP on dried blood without DNA extraction.

We have developed a simple, labor-saving, inexpensive, and rapid single nucleotide polymorphism (SNP) genotyping method that works directly on whole human blood. This single-tube genotyping method was used to successfully and reliably genotype ADH1B and ALDH2 polymorphisms without DNA isolation using a 1.2-mm disc of dried blood and the KOD FX PCR enzyme kit.

Single-tube genotyping from a human hair root by direct PCR.

We have developed a simple, labor-saving, inexpensive and rapid SNP genotyping method that directly uses a human hair root as the template. This single-tube genotyping method was used to successfully and reliably genotype the ADH1B and ALDH2 polymorphisms using a hair root (without DNA isolation) and the polymerase chain reaction (PCR) enzyme kit KOD FX. Since the DNA extraction step was eliminated, the possibility of sample contamination was considerably decreased.

All-in-one tube method for quantitative gene expression analysis in oligo-dT(30) immobilized PCR tube coated with MPC polymer.

In this report, we have developed a novel quantitative RT-PCR protocol in which the procedure including mRNA purification can be performed in an all-in-one tube. To simplify gene expression analysis, oligo-dT(30) immobilized PCR tubes were used serially to capture mRNA, synthesize solid-phase cDNA, and amplify specific genes. The immobilized oligo-dT(30) can efficiently capture mRNA directly from crude human cell lysates.

A comparative analysis of the transcriptome and signal pathways in hepatic differentiation of human adipose mesenchymal stem cells.

The specific features of the plasticity of adult stem cells are largely unknown. Recently, we demonstrated the hepatic differentiation of human adipose tissue-derived mesenchymal stem cells (AT-MSCs). To identify the genes responsible for hepatic differentiation, we examined the gene expression profiles of AT-MSC-derived hepatocytes (AT-MSC-Hepa) using several microarray methods.

Multiple primer extension by DNA polymerase on a novel plastic DNA array coated with a biocompatible polymer.

DNA microarrays are routinely used to monitor gene expression profiling and single nucleotide polymorphisms (SNPs). However, for practically useful high performance, the detection sensitivity is still not adequate, leaving low expression genes undetected. To resolve this issue, we have developed a new plastic S-BIO PrimeSurface with a biocompatible polymer; its surface chemistry offers an extraordinarily stable thermal property for a lack of pre-activated glass slide surface.

Psychophysiological stress-regulated gene expression in mice.

Eight genes showed significant changes in expression in mice under psychophysiological stress provided by cage-restraint and water-immersion. The transcription level of most of these genes was affected in all the tissues analyzed, and some of them were responsive genes in several different stress systems. Peculiarly, the expression level of one gene, cdc2-like kinase 1 (CLK1), was reduced only in the brain, while the balance of partially- and alternatively-spliced CLK1 mRNA species changed in all the tissues including the brain.