PCR Used to Find Plasmid Backbone Fragments in the Products of hiTAIL-PCR
Received date: 2016-11-11
Accepted date: 2017-04-17
Online published: 2017-04-17
T-DNA mutants are important resources for research of gene function. High-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) is widely used for cloning the flanking sequence near the T-DNA insertion sites. However, we found that some cloned flanking fragments in hiTAIL-PCR products corresponded not to the host genomic DNA but to the plasmid backbone DNA. In this study, with a control of the RB-S4/AC1 or LB-A4/AC1 product, we amplified PCR fragments from the plasmid backbone DNA. By excluding them from further analysis, we amplified fragments from the unknown genomic DNA more effectively. Meanwhile, by adjusting the PCR programs, the whole PCR time was greatly shortened. In cloning the flanking sequence of Arabidopsis thaliana T-DNA mutant drf1, our method with hiTAIL-PCR reduced the total 22 DNA bands required for further checking to 4 bands, which improved the efficiency by 81.8%.
Key words： hiTAIL-PCR; T-DNA mutant; flanking sequence; plasmid backbone; cloning
Yuan Cao, Yun Yang, Huaquan Xu, Yang Liu, Danyang Wan . PCR Used to Find Plasmid Backbone Fragments in the Products of hiTAIL-PCR[J]. Chinese Bulletin of Botany, 2018 , 53(1) : 104 -109 . DOI: 10.11983/CBB16216
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