Takara Bio Inc. (President and CEO : Ikunoshin Kato, Ph.D.) releases kits that easily differentiate the genetic polymorphism that is responsible for different levels of susceptibility to alcohol and cigarette based on ICAN^TM and UCAN^TM gene amplification methods that Takara Bio developed, starting December 16, 2002. These kits target the human aldehyde dehydrogenase gene responsible for alcohol metabolism, and the human glutathione-S-transferase M1 gene responsible for metabolic degradation of various materials including carcinogenic substances derived from cigarette smoke, and other drugs.

Differences in susceptibility to alcohol are caused by differences in genes coding enzymes responsible for the metabolism of alcohol. Aldehyde dehydrogenase (ALDH2) metabolically degrades and detoxifies acetaldehyde, a poisonous substance generated from the metabolic degradation of alcohol in the body. On the gene of ALDH2, there exists the Single Nucleotide Polymorphism (SNP) . Differences in alcohol susceptibility are caused by the different SNP characteristics. For example, the person who has an inactive type of ALDH2 cannot effectively metabolize acetaldehyde. As a result, the level of acetaldehyde in blood rises and it may cause headaches and nausea. Takara Bio Research lab has developed two kinds of kits that can discriminate the genetic polymorphism of ALDH2.

The first kit can determine genetically SNP using UCAN^TM, which is novel SNP typing methodology. In the protocol using this kit, genotyping of the SNP is done by amplifying SNP-carrying genes using the UCAN method and then colorimetrically detecting the degree of the amplification with a probe that selectively hybridizes the amplified regions of the gene. This kit dose not need a special device, and the operations can be finished within 3 hours.

A characteristic feature of the second kit is the use of a DNA-RNA-DNA chimera oligonucleotide (DRD) probe, the RNA region of which corresponds to the SNP position of the gene. Since this probe is modified with a fluorescent agent and a quenching agent on each end or nearby, no fluorescence is generated as long as the region remains intact. Complementary DNA is amplified as the course of reaction progress, and if the probe and the amplified fragments completely match, RNaseH breaks the RNA region (SNP position) of the probe, and fluorescence signal produces. In addition, because the fluorescence agents that generate different colors are used for the two types of the probe sequence that correspond to different SNP types, the SNP genotyping is possible by detecting the fluorescence color. Since the ICAN^TM reaction and detection reaction progress simultaneously and identification is possible by the fluorescence color-based differentiation, the SNP genotyping can be carried out quickly without complicated procedure

Many of the chemical substances taken into the body are metabolized by enzymes in the liver, among which glutathione-S-transferase M1 (GSTM1) is an important member that detoxifies chemicals by adding glutathione to them. Despite the physiological importance of this enzyme, there are people who lack the gene that codes GSTM1. Such people may suffer from higher risk of acquiring cancers, or experience various adverse side effects from drugs. Because this enzyme is responsible for detoxifying benzpyrene derivatives that are carcinogenic products generated from smoking, the person lacking the gene have a higher risk of lung cancer if they are exposed to cigarette smoke.

This kit can determine genetically the polymorphism of this deficiency by amplifying the corresponding genes with ICAN^TM primers on a part of the deficient region (ICAN method) and then colorimetrically detecting the amplified genes on a microplate with a probe that specifically hybridizes the amplified regions. Because this kit uses Multiplex ICAN^TM, which is capable of identifying the presence of DNA in the sample and the success of the amplification reaction through beta-globin gene amplification, it can detect false negative reactions caused by erroneous reactions, inhibited amplification, or faulty preparation of DNA.

This kit can quickly and easily perform all the genotyping operations from amplification to detection in less than 3 hours without requiring specialized equipment.

These kits are suitable to not only blood DNA samples, but also oral mucous membrane DNA that is easily collectable. Because no blood drawing is involved in the latter case, the tests can be performed very safely and easily.

The research of the relationship between genetic polymorphism and the physiological characteristics of individuals is progressing with accelerating speed. This makes spurring demands for methodologies that enhance the diagnosis of pathopoiesis and the choice of treatment and preventive method. In order to put the results of this research to practical use, a quick and easy genotyping method is in high demand.

The genotyping kit utilizes UCAN and ICAN methods that Takara Bio has developed, which can be applied to all genetic polymorphisms found among humans. Takara Bio plans to license the technology to outside parties including pharmaceutical companies, and plans to provide the necessary services for deploying the technology. This includes designing the genotyping kits, and assembling the kits to meet users' demands.

This article is translated from news in Japanese for your convenience.