一个高效的非自然基对用于PCR放大.
Ichiro Hirao1, Tsuneo Mitsui, Michiko Kimoto
1Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. ihirao@riken.jp
Journal of the American Chemical Society
|November 22, 2007
概括
研究人员开发了一种新的非自然基对Ds-Pn,用于DNA放大. 这种工程遗传系统在PCR中表现出高效率和选择性,使生物技术应用得以扩大.
科学领域:
- 生物技术是生物技术.
- 合成生物学 合成生物学
- 分子生物学分子生物学
背景情况:
- 使用非自然基对扩展遗传字母是生物技术的一个关键进步.
- 以前的非自然基对在DNA复制和放大过程中在效率和选择性方面存在局限性.
研究的目的:
- 开发和表征一种新的非自然基对,用于增强DNA放大.
- 评估PCR中新型Ds-Pn基对的效率和保真性.
主要方法:
- 开发了一种新的非自然基对:7-(2-thienyl) imidazo[4,5-b]pyridine (Ds) 和2-nitropyrrole (Pn).
- 使用特定基质混合物 (Ds的胺三酸盐和天然三酸盐) 与Vent DNA聚合酶进行PCR放大.
- 评估Pn与Ds对配的选择性,而不是其他基,特别是氨酸 (A).
主要成果:
- 不自然的Ds-Pn基对通过PCR在DNA放大中表现出高效率和选择性.
- 在Pn中,基组有效地防止了与腺素 (A) 的错误配对.
- 使用Ds-Pn对进行PCR放大,在20个循环后,在非自然的基对位点产生了约1%的低突变率.
结论:
- 新的Ds-Pn非自然基对在DNA放大中有效地起作用.
- 这种工程基因对为扩展基因系统在基于DNA的生物技术中提供了有价值的工具.
- Ds-Pn对的高保真性和效率为新的生物技术应用铺平了道路.
相关概念视频
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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
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PCR - Polymerase Chain Reaction
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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...
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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
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