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J Cancer. 2015 May 25;6(7):632-5. doi: 10.7150/jca.11779. eCollection 2015.

Restriction-based Multiple-fragment Assembly Strategy to Avoid Random Mutation during Long cDNA Cloning.

Author information

1
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.

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.

KEYWORDS:

CDS; PCR; cloning strategy; endonuclease.; ligation

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