Ligation Based Assembly and Polymerase Chain Reaction-Based Assembly for Extraordinary Adenine/Thymine Rich DNA
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Extraordinary adenine/thymine rich DNA has a low complexity resulting in the occurrence of many short repetitive sequences or longer imperfect repeats, consequently hindering the gene synthesis process. This report describes the rapid synthesis of a DNA fragment with guanine/cytosine content of 11.8% using ligation based assembly and polymerase chain reaction-based assembly respectively, via the use of multiple strategies addressing the adenine/thymine rich nature of the fragment. Sequences can be simply chopped and assembled without Tm optimization. Smaller amount of ligation products as templates in Interference Free polymerase chain reaction yielded markedly more than or equal amount of subassembly products as using larger amount of ligation products, and longer extension time was required for successful subassembly. Longer time at low annealing temperature also had obvious effects on polymerase chain reaction amplifications. No full-length products could be generated without initial oligonucleotide sub-pooling for both approaches. The chemical synthesis of extraordinary adenine/thymine rich DNA could enable researchers to assemble synthetic modules, to study and to access the repetitive DNA such as heterochromatin regions which harbor important functional elements.
KeywordsExtraordinary adenine/thymine rich DNA Low complexity Ligation based assembly Polymerase chain reaction-based assembly
This work was supported by the Guangdong Natural Science Foundation (S2011010004264), Guangdong Science and Technology Program (No. 2008B020100001, 2016B020204001), Open Fund of MOE Key Laboratory of Aquatic Product Safety, Foreign Expert Program at Sun Yat-sen University, Open Fund of Laboratory (20160215) and 2016 Key Project Budget at Sun Yat-sen University, and The National Natural Science Foundation of China (J1310025, 21601209, 30370799). The authors thank Dr. Yuchuan Wang, Ms Jing Li, Mr. Xiang Zhu, Ms Fan Yang, Mr. Xingqiang Lai and Ms Cui Yang for technical help. The authors are grateful to Dr. Weiguo Cao and Ms Yan Shi for editorial assistance, and anonymous reviewer for valuable suggestions.
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Conflict of interest
The authors declare that they have no conflict of interest.
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