CyDNA:通过一个进化的聚合酶合成和复制高度Cy染料替代的DNA
Nicola Ramsay1, Ann-Sofie Jemth, Anthony Brown
1MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, United Kingdom.
Journal of the American Chemical Society
|March 19, 2010
概括
研究人员开发了一种方法,通过用染料标记的版本替换DNA基来创建光DNA (CyDNA). 这种鲜的颜色,高度光的DNA在微阵列和微流体学中改变了特性和潜在的应用.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 材料科学 材料科学 材料科学
背景情况:
- DNA 作为遗传载体和超分子支架.
- 目前的DNA合成方法在结合各种替代剂方面存在局限性.
研究的目的:
- 开发一种用数百种替代品合成和复制DNA的方法.
- 为了创建一个颜色鲜和高度光的DNA分子,具有改变的属性.
主要方法:
- 通过短补丁区分自我复制 (spCSR) 利用聚合酶功能的定向进化.
- 使用光标记的脱氧核酸三酸盐 (Cy3-dCTP和Cy5-dCTP) 作为基质.
- 隔离了一种能够进行PCR增强DNA的聚合酶,其中所有dC基都被光标签所替代.
主要成果:
- 成功合成了高达1kb的双链DNA片段,其中所有dC基都被Cy3-或Cy5-dC取代,称为"CyDNA".
- 赛DNA呈现出明亮的光,改变了物理化学特性 (例如,减少了结合,内核酶抵抗) 和增加了直径.
- 在微阵列和微流体应用中证明了信号增强的潜力.
结论:
- 赛DNA代表了一种新的DNA变体,具有显著的特性变化和增强的光.
- 这一进步朝着编码合成具有可编程序列和特性的基于DNA的聚合物的目标迈进.
- 在生物技术和材料科学方面,CyDNA具有先进应用的潜力.
相关概念视频
02:10Translesion DNA Polymerases
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TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
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02:31Homologous Recombination
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02:40DNA Replication
DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied. After replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
Replication in Prokaryotes
DNA replication uses a large number of...
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01:31Proofreading
Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore, it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
Errors During Replication are Corrected by the DNA Polymerase Enzyme
Errors During Replication are Corrected by the DNA Polymerase Enzyme
01:43Proofreading
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01:59Lagging Strand Synthesis
During replication, the complementary strands in double-stranded DNA are synthesized at different rates. Replication first begins on the leading strand. Replication starts later, occurs more slowly, and proceeds discontinuously on the lagging strand.
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There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...