National Academy Science Letters

, Volume 41, Issue 4, pp 207–210 | Cite as

The Length Limit of 5′ Nucleotide Additions to PCR Primers

  • Chang-En TianEmail author
  • Tao-Wen Hong
  • Yu-Ping Zhou
  • Qiong-Hua Chen
  • Xiao-Ling Huang
  • Xiao-Yi Guo
Short Communication


The addition of nucleotides to polymerase chain reaction (PCR) primers has become a widely used technique to facilitate the cloning of PCR products. A long fragment mismatched with template, such as an epitope-encoding sequence, needs to be added to the 5′ end of the primer for identification and purification of the expressed protein. However, the length limit of added sequences has not yet been determined. Under our condition, the maximum length for 5′ nucleotide additions was found to be 108, when a fully matched forwards primer and partially matched reverse primer contained 44.4–55.6% GC in the matched region with template are used. The results shown here are very useful for scientists who want to add a long mismatched sequence with template, such as an epitop/a signal peptide encoding sequence and/or the multiple cloning sites.


5′ nucleotide additions PCR Primer The length limit 



This work was supported by the National Natural Science Foundation of China (Nos. 31170204 and 31470014) and the Science and Technology Planning Project of the Education Bureau of Guangzhou Municipality (Nos. 12A001G, 1201420546 and 8151009101000013).


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Copyright information

© The National Academy of Sciences, India 2018

Authors and Affiliations

  • Chang-En Tian
    • 1
    Email author
  • Tao-Wen Hong
    • 1
  • Yu-Ping Zhou
    • 1
  • Qiong-Hua Chen
    • 1
  • Xiao-Ling Huang
    • 1
  • Xiao-Yi Guo
    • 1
  1. 1.School of Life SciencesGuangzhou UniversityGuangzhouChina

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