基因消除PCR

       基因消除PCR是基于限制性酶的PCR反向过程的一种去除非目标基因的新技术，简称为R-PCR (Removing PCR, Restriction PCR, or Reverse PCR)。在PCR反应中，每一循环目标基因得到了扩增；在R-PCR反应中，每一循环非目标基因得到了去除。目前，研究基因差异表达谱主要是通过高通量二代测序（RNA-seq），生物芯片(microarrays)，抑制差减杂交（Subtractive Suppression Hybridization，SSH）等完成的。这些方法各有优缺点，且没有一种方法可以简易高效地从基因群体中去除非目标基因。PCR及限制性酶切都是比较可靠高效的技术。限制性酶ApeKI是NEB公司的一种time-saver酶且能与PCR反应缓冲液兼容。根据这些技术特点华中农业大学的董五辈教授及中国农业科学院的王国英教授所领导的两个课题组合作共同建立了R-PCR技术，文章发表在本周出版的Scientific Reports上，桓娇娇和万康康是共同第一作者。R-PCR可应用于研究差异表达的基因及特异性的基因组DNA，从而克隆目标基因。R-PCR可替代的技术包括抑制差减杂交（SSH），差异显示（DD）等。 

A reverse process of PCR has been developed which can eliminate undesired genes cycle by cycle

http://www.sciguru.com/newsitem/16371/reverse-process-pcr-eliminate-unwanted-genes-cycle-cycle-new-invention

Polymerase chain reaction (PCR) which is a technique used to amplify many copies of a desired DNA fragment, cycle by cycle, was developed by Kary B. Mullis in 1983. After 30 years, a reverse process of PCR, removing PCR (R-PCR), has been developed which can eliminate undesired DNA fragments, cycle by cycle. If researchers want to amplify their desired genes, they can use PCR, whereas, if researchers want to remove their undesired genes, they can use the R-PCR.

In life sciences, researchers always need to know which genes are present or absent and which genes are induced or repressed. Although the next generation high-throughput sequencing technology is available, it is still expensive, and post-sequencing data analysis is complicated. PCR is a fast, inexpensive, and widely used technique to amplify desired genes. However, how undesired genes are removed from a gene pool that includes both desired and undesired genes remains a challenge. Researchers from Huazhong Agricultural University and Chinese Academy of Agricultural Sciences have developed the novel R-PCR technique which can eliminate the undesired genes.

The R-PCR technique, which has been published today, 29 July, in Scientific Reports by Nature Publishing Group, is a counterpart of PCR. However, in each R-PCR cycle, genes are removed, whereas, in each cycle of regular PCR, genes are amplified. The R-PCR represents an alternative to several existing techniques for the identification of differentially expressed genes, including the RNA-seq. In the future, drivers in the R-PCR system may be made shorter and become commercially available for specific samples or species. Jiaojiao Huan and Kangkang Wan are the co-first authors in the publication. Other authors on the paper are Yunjun Liu, Wubei Dong, and Guoying Wang.

Major principle of the R-PCR is to let drivers kill undesired genes in a gene pool which includes both desired and undesired genes. The desired genes can survive in R-PCR reactions because a mismatched base pair protects them from digestion by ApeKI restriction enzyme and there is no driver to kill them. The researchers found that after the R-PCR reactions, most undesired genes, including very abundant genes, became undetectable. The R-PCR is an easy and cost-efficient method to eliminate undesired genes and clone desired genes.

This research led by Prof. Wubei Dong fromHuazhongAgriculturalUniversityand Prof. Guoying Wang fromChineseAcademyof Agricultural Sciences was supported by the National Basic Research Program of China (2009CB118404) and the National Major Project for Transgenic Organism Breeding inChina(2011ZX08003-001).

Journal Reference:

Jiaojiao Huan, Kangkang Wan, Yunjun Liu, Wubei Dong* & Guoying Wang. Removing PCR for the elimination of undesired DNA fragments cycle by cycle. Scientific Reports 3, 2303; DOI:10.1038/srep02303 (2013).

http://www.nature.com/srep/2013/130729/srep02303/full/srep02303.html