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中国生物工程杂志

China Biotechnology
China Biotechnology  2017, Vol. 37 Issue (2): 74-80    DOI: 10.13523/j.cb.20170211
    
Establishment of the Single-cell PCR System and Its Application in the Target-activity Detection of CRISPR/Cas9 System
XU Zhen-yu1,2, REN Hong-yan2, BI Yan-zhen2, ZHENG Xin-min2, LI Li2, ZHANG Jia-lan1
1. College of Animal Sciences, Yangtze University, Jingzhou 434025, China;
2. Key Laboratory of Animal Embryo Engineering and Molecular Breeding of Hubei Province, Institute of Animal and Veterinary Sciences, Hubei Academy of Agricultural Sciences, Wuhan 430064, China
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Abstract  

Single-cell PCR uses the DNA or RNA contained in a single cell as template for the amplification.The quality of the template is one of the key factors for the success of the reaction.Three different proteinase K cell lysate buffers (SDS cell lysate buffer,NP-40 cell lysate buffer and Tween-20 cell lysate buffer) were used to digest the matured oocytes of porcine in vitro.The product of digestion was used as the template for single-cell PCR amplification directly.The results showed that the quality of template processed by NP-40 cell lysates buffer was the best,with a high amplification efficiency of 95%.The amplification efficiency of PCR with template produced by Tween-20 cell lysates buffer was about 65%;while there was no positive bands were detected using the SDS cell lysates Buffer.Combined the single-cell PCR with the microinjection techniques,and it has been used to detect the activity of CRISPR-Cas9 target in vitro.The results showed that this detection method has a high efficiency and stability.



Key wordsCell lysates Buffer      Activity detection of target      Genetic modified animal      Single-cell PCR     
Received: 03 August 2016      Published: 25 February 2017
ZTFLH:  Q819  
Cite this article:

XU Zhen-yu, REN Hong-yan, BI Yan-zhen, ZHENG Xin-min, LI Li, ZHANG Jia-lan. Establishment of the Single-cell PCR System and Its Application in the Target-activity Detection of CRISPR/Cas9 System. China Biotechnology, 2017, 37(2): 74-80.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20170211     OR     https://manu60.magtech.com.cn/biotech/Y2017/V37/I2/74

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