Molecular Designing of amiRNA and Its synthesis Strategy in vitro

doi:Q52

China Biotechnology

2010, Vol. 30

Issue (09): 92-97 DOI: Q52

Molecular Designing of amiRNA and Its synthesis Strategy in vitro

YE Mei-xia,CUI Dong-qing,LI Hao,WANG Jing-cheng,LIU Jun-mei,ZHANG Zhi-yi,AN Xin-min

National Engineering Laboratory for Tree Breeding, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education， The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of State Forestry Administration, Beijing Forestry University, Beijing 100083, China

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Abstract

Detailedly described several reliable methods for constructing amiRNA , which was based on WMD3 (Web MicroRNA Designer 3), including molecular designing and flexible synthesis strategy in vitro. When designed online, four steps were needed sequentially: inputting target sequence(s), screening candidate amiRNAs, gaining four oligonucleotides carrying amiRNA-related sequence and other proper sequences that flanked nature miRNA. For overlapping PCR synthesis strategy, amiRNA can be amplified using these four oligonucleotides and two primers designed based on plasmid. Uracil-excision based strategy could also be employed to generate amiRNA, while primers need altering. Specific primer-annealing based method was equally represented here, which may guarantee the synthesis of amiRNA in one-step-PCR. With appropriate restriction sites, the synthesized amiRNA expression cassette can be cloned into corresponding sites of destination vector. It is foreseeable that such scientificness of molecular designing principle and the accuracy of synthesis strategy would aid greatly in gene functional analysis, promising a profound impact on life science.

Key words： WMD3 amiRNA Molecular design Synthesis strategy

Received: 19 April 2010 Published: 25 August 2010

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	XIE Mei-Xia
	CUI Dong-Qing
	LI Hao
	WANG Jing-Cheng
	LIU Jun-Mei
	ZHANG Zhi-Yi
	AN Xin-Min

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XIE Mei-Xia, CUI Dong-Qing, LI Hao, WANG Jing-Cheng, LIU Jun-Mei, ZHANG Zhi-Yi, AN Xin-Min. Molecular Designing of amiRNA and Its synthesis Strategy in vitro. China Biotechnology, 2010, 30(09): 92-97.

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https://manu60.magtech.com.cn/biotech/Q52 OR https://manu60.magtech.com.cn/biotech/Y2010/V30/I09/92

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