权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Enzymatic Synthesis of Modified DNAs by PCR, and Their Application

PCR 酶法合成修饰 DNA 及其应用

基本信息

批准号：
13132202
负责人：
SAWAI Hiroaki
金额：
$ 24.32万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13132202/
关键词：
Nucleic Acid Modified DNA DNA polymerase Modified Substrate PCR Enzymatic Reaction Molecular Recognition Aptamer DNA 生体機能利用糖鎖機能性DNA DNAアプタマー DNA触媒試験管内選択

项目摘要

If modified DNA can be prepared enzymatically by PCR from the substrate analogue using DNA polymerase, The resulting modified DNA could be used as a DNA probe by attachment of a reporter group, and as a DNA catalyst or as a DNA aptamer by in vitro selection. We prepared thymidine analogues bearing various functional groups at the C5 position, and examined whether they can be accepted as a substrate for the DNA polymerase. The functional groups studied in this research are an amino group, a carboxyl group, chelating groups, guanidine, imidazole, pyridine, various amino acids, fluorescein, acridone and sugars. We found that KOD Dash and Vent(exo-) DNA polymerases can incorporate many of these modified thymidine analogues and that the corresponding modified DNA can be obtained by PCR. On the other hand, no other DNA polymerases such as Taq DNA polymerase can accept these modified substrates. The resulting modified DNA shows no mutation at the modified portion. Similarly, cytidine analogues bearing several functional groups at C5 position can work as a substrate for PCR using the DNA polymerases. We also confirmed that modified DNA could be prepared by PCR from some modified adenine nucleotides. Further, combination use of modified thymidine, cytidine and adenine nucleotides for the PCR yielded the corresponding multi-modified DNA. We applied the modified DNA synthesis by PCR for the production of DNA aptamers using an in vitro selection technique. Modified DNA aptamers that bind specifically with sialllylactose that is present in a surface of cells, glutamic acid, a drug thalidomide or secondary and ternary structure of DNA have been developed. The sequence, secondary structure and binding affinity of the aptamers to the target compound have been studied to characterize the aptamers.

如果用DNA聚合酶从底物类似物中用酶法从底物中制备DNA，那么得到的修饰DNA可以通过连接报告基团作为DNA探针，也可以通过体外选择作为DNA催化剂或DNA适配子。我们制备了在C5位带有不同功能基团的胸苷类似物，并检测了它们是否可以作为DNA聚合酶的底物。本研究所研究的官能团包括氨基、羧基、螯合基团、胍、咪唑、吡啶、各种氨基酸、荧光素、吖啶酮和糖。我们发现KOD、DASH和Vent(exo-)DNA聚合酶可以结合许多这些修饰的胸苷类似物，并且相应的修饰DNA可以通过聚合酶链式反应获得。另一方面，其他DNA聚合酶如Taq DNA聚合酶都不能接受这些修饰的底物。由此得到的修饰DNA在修饰部分没有显示突变。类似地，在C5位带有几个官能团的胞苷类似物可以作为使用DNA聚合酶进行聚合酶链式反应的底物。我们还证实了一些修饰的腺嘌呤核苷酸可以通过聚合酶链式反应来制备修饰的DNA。此外，将修饰的胸腺嘧啶核苷、胞苷和腺嘌呤核苷酸组合用于聚合酶链式反应可产生相应的多修饰DNA。我们将改进的DNA合成方法应用于DNA适配子的体外筛选技术。已开发出与存在于细胞表面的唾液酸基乳糖、谷氨酸、药物沙利度胺或DNA的二级和三元结构特异性结合的修饰DNA适配子。研究了适配子的序列、二级结构和与目标化合物的结合亲和力，以表征适配子。