扩大DNA的化学特征以进行体外选择
Jonathan D Vaught1, Chris Bock, Jeff Carter
1Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Journal of the American Chemical Society
|March 6, 2010
概括
改性脱氧核酸三酸盐 (dUTP) 衍生物能够成功地对难以达到的蛋白质标进行DNA吸收体选择. 这些修改后的dUTPs促进了高亲和度的阿巴特马分离,克服了TNFRSF9.9在体外选择的先前限制.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 合成化学 合成化学
背景情况:
- 试管体内选择是一种用于发现DNA体的强大方法.
- 某些蛋白质标,如TNFRSF9,已经耐火于使用标准脱氧核酸的传统体外选择方法.
- 改性脱氧核酸三酸盐 (dUTP) 衍生物具有克服这些限制的潜力.
研究的目的:
- 合成和评估用于体外选择的新型5位修饰dUTP衍生物.
- 评估这些修改后的dUTP与酶性DNA合成的兼容性,包括PCR和原料扩展.
- 为了确定修改后的dUTP是否可以使DNA体对以前难以处理的目标TNFRSF9.9进行选择.
主要方法:
- 合成了六种新型的5位修饰dUTP衍生物,其中包括一种胺链接.
- 测试六种商业DNA聚合酶在PCR和原料延伸反应中纳入这些修饰的dUTP的能力.
- 使用对TNFRSF9的修改DNA库进行体外选择实验,将结果与标准脱氧核酸三酸盐 (TTP) 进行比较.
主要成果:
- 修改后的dUTPs与标准PCR条件不兼容,但在原始延伸反应中成功被纳入.
- KOD XL和D. Vent DNA聚合酶在合成具有修改dUTP的全长原料延伸产品方面表现出高效率.
- 使用改造的DNA图书馆进行体外选择,为TNFRSF9产生了第一个高亲和度DNA受体,TNFRSF9是以前对受体选择有抗性的受体.
结论:
- 新的5位修改dUTP衍生物可以有效地用于原料延伸反应.
- 这些修改后的dUTPs能够成功地在体外选择DNA体,以对抗具有挑战性的蛋白质标,如TNFRSF9.
- 开发的修改后的dUTPs代表了对以前难以处理的目标的aptamer发现的重大进步.
相关概念视频
01:24DNA Isolation
DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
00:57Antibiotic Selection
Overview
01:16In vitro Mutagenesis
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
01:20The Central Dogma
The central dogma explains the flow of genetic information from DNA nucleotides to the amino acid sequence of proteins.
RNA is the Missing Link Between DNA and Proteins
In the early 1900s, scientists discovered that DNA stores all the information needed for cellular functions and that proteins perform most of these functions. However, the mechanisms of converting genetic information into functional proteins remained unknown for many years. Initially, it was believed that a single gene is...
RNA is the Missing Link Between DNA and Proteins
In the early 1900s, scientists discovered that DNA stores all the information needed for cellular functions and that proteins perform most of these functions. However, the mechanisms of converting genetic information into functional proteins remained unknown for many years. Initially, it was believed that a single gene is...