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Development of a Practical SNPs Typing Method by Using Elactro-ActiveArtificial DNAs

使用电活性人工 DNA 开发实用的 SNP 分型方法

基本信息

批准号：
18350082
负责人：
INOUYE Masahiko
金额：
$ 10.77万
依托单位：
University of Toyama
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18350082/
关键词：
DNA Probe SNP Ferrocene Au Electrode Electrochemistry

项目摘要

Most human DNA polymorphisms are single-nucleotide polymorphisms (SNPs), which critically relate to risks of various common diseases and differences in drug response. The clinical importance of detecting SNPs has inspired investigations into numerous assay systems that can discriminate SNP alleles from normal ones. However, the existing systems are not satisfactory, particularly when applied to a hand-held SNPs sensor, which is useful for end users. The ultimate assay protocol must allow end users to only need to directly add the sample that contains the PCR-amplified target DNAs onto a low-cost chip in a portable device.We have reported an electrochemical detection of complementary DNAs that is based on hole transport with ferrocene-p-conjugated DNA probes. This method allows a reagentless discrimination of label-free DNA from their SNPs with quasi "on/off" digital response. Nevertheless, to meet the criteria of the above-mentioned assay protocol, our method should thoroughly be remad … More e in terms of synthetic cost for the electroactive pseudonucleoside, in situ hybridization of target DNAs on premodified electrode with probes, and applicability to the longer target DNAs than are produced by common PCR. Here we investigated new ferrocene-p-conjugated DNA probes that almost satisfy the requirements for realizing a user-friendly SNP sensor that is highly compatible with PCR on-chip technologies.We have prepared new DNA probes of various structures and examined their SNP discrimination abilities for target sequences of many transition and transversion SNPs of clinical importance, with the goal of the above-mentioned practical use in mind. As a result, we have developed a new electrochemical SNP typing system composed of synthetically feasible and versatile, cheap electro-active DNA probes. This system is expected to meet the criteria of the assay protocol required by end users majoring in clinical medicines. Further improvements of our methodology are in progress for realizing an ideal SNPs sensor. Less

大多数人类DNA多态性是单核苷酸多态性（SNPs），它与各种常见疾病的风险和药物反应的差异密切相关。检测SNP的临床重要性激发了对许多可以区分SNP等位基因与正常等位基因的测定系统的研究。然而，现有的系统并不令人满意，特别是当应用于手持SNP传感器时，这对最终用户是有用的。最终的检测方案必须允许最终用户只需要直接添加包含PCR扩增的目标DNAs的样品到便携式设备中的低成本chip上。我们已经报道了互补DNAs的电化学检测，该检测基于与二茂铁-p-缀合的DNA探针的空穴传输。该方法允许无试剂区分无标记DNA与它们的SNP，具有准“开/关”数字响应。然而，为了满足上述测定方案的标准，我们的方法应彻底改进， ...更多信息在电活性核苷的合成成本、靶DNA在预修饰电极上与探针的原位杂交以及对比普通PCR产生的靶DNA更长的靶DNA的适用性方面，为了实现与芯片上PCR技术高度兼容的SNP传感器，我们研究了几乎满足要求的新型二茂铁-p-共轭DNA探针。我们制备了各种结构的新型DNA探针，并以上述实际应用为目的，研究了它们对许多临床重要的转换和颠换SNP的靶序列的SNP识别能力。因此，我们开发了一种新的电化学SNP分型系统，该系统由综合可行且通用的廉价电活性DNA探针组成。预期该系统符合临床医学专业最终用户要求的测定方案标准。我们的方法的进一步改进正在进行中，以实现理想的SNP传感器。少