title: Restriction-based Multiple-fragment Assembly Strategy to Avoid Random Mutation during Long cDNA Cloning.
abstract: Long fragment cloning is a challenge for its difficulty in accurate amplifying and tendency to get unwanted mutation. Here we discuss Restriction-based Multiple-fragment Assembly Strategy's advantages and limitations. In this strategy, rather than PCR amplifying the entire coding sequence (CDS) at one time, we amplified and sequenced smaller fragments which are shorter than 1.5kb spanning the CDS. After that, the sequence-proved fragments were assembled by digestion-ligation cloning to the target vector. We test its universality in our script programmed in Python. Our data shows that, among the entire human and mouse CDS, at least 70% of long CDS cloning will benefit from this strategy.
authors.affiliation: 1. Key Laboratory of Protein Chemistry and Developmental Biology of Education Ministry of China, College of Life Sciences, Hunan Normal University, Changsha 410081, China; ; 2. The Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486), Changsha 410081, China.
authors.forename: Shang
authors.initials: S
authors.lastname: Wang
authors.affiliation: 1. Key Laboratory of Protein Chemistry and Developmental Biology of Education Ministry of China, College of Life Sciences, Hunan Normal University, Changsha 410081, China; ; 2. The Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486), Changsha 410081, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Chen
authors.affiliation: 1. Key Laboratory of Protein Chemistry and Developmental Biology of Education Ministry of China, College of Life Sciences, Hunan Normal University, Changsha 410081, China; ; 2. The Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486), Changsha 410081, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhang
authors.affiliation: 1. Key Laboratory of Protein Chemistry and Developmental Biology of Education Ministry of China, College of Life Sciences, Hunan Normal University, Changsha 410081, China; ; 2. The Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486), Changsha 410081, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Jiao
authors.affiliation: 1. Key Laboratory of Protein Chemistry and Developmental Biology of Education Ministry of China, College of Life Sciences, Hunan Normal University, Changsha 410081, China; ; 2. The Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486), Changsha 410081, China.
authors.forename: Lihua
authors.initials: L
authors.lastname: Mo
authors.affiliation: 1. Key Laboratory of Protein Chemistry and Developmental Biology of Education Ministry of China, College of Life Sciences, Hunan Normal University, Changsha 410081, China; ; 2. The Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486), Changsha 410081, China.
authors.forename: Xiaoxu
authors.initials: X
authors.lastname: Yang
authors.affiliation: 1. Key Laboratory of Protein Chemistry and Developmental Biology of Education Ministry of China, College of Life Sciences, Hunan Normal University, Changsha 410081, China; ; 2. The Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486), Changsha 410081, China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Hu
authors.affiliation: 1. Key Laboratory of Protein Chemistry and Developmental Biology of Education Ministry of China, College of Life Sciences, Hunan Normal University, Changsha 410081, China; ; 2. The Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486), Changsha 410081, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.affiliation: 1. Key Laboratory of Protein Chemistry and Developmental Biology of Education Ministry of China, College of Life Sciences, Hunan Normal University, Changsha 410081, China; ; 2. The Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486), Changsha 410081, China.
authors.forename: Chenxi
authors.initials: C
authors.lastname: Wei
authors.affiliation: 1. Key Laboratory of Protein Chemistry and Developmental Biology of Education Ministry of China, College of Life Sciences, Hunan Normal University, Changsha 410081, China; ; 2. The Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486), Changsha 410081, China.
authors.forename: Shuanglin
authors.initials: S
authors.lastname: Xiang
keywords: CDS; PCR; cloning strategy; endonuclease.; ligation