The Length Limit of 5′ Nucleotide Additions to PCR Primers
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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.
Keywords5′ 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).
- 7.Mead DA, Pey NK, Hernstadt C, Marcil RA, Smith LM (1991) A universal method for the direct cloning of PCR amplified nucleic acid. Biotechnol 9:657–663Google Scholar
- 8.Dillon PJ, Rosen CA (1993) Use of polymerase chain reaction for the rapid construction of synthetic genes. In: White BA (ed) Methods in molecular biology, vol 15. Humana Press Inc, Totowa, pp 263–268Google Scholar
- 11.Su CT, Schonbach C, Ck K (2013) Molecular docking analysis of 2009-H1N1 and 2004-H5N1 influenza virus HLA-B* 4405-restricted HA epitope candidates: implications for TCR cross-recognition and vaccine development. BMC Bioinform 14(S21):347–360Google Scholar
- 13.Tian C, Muto H, Higuchi K, Matamura T, Tatematsu K, Koshiba T, Yamamoto KT (2004) Disruption and overexpression of Auxin Response Factor 8 gene of Arabidopsis affect hypocotyl elongation and root growth habit, indicating its possible involvement in auxin homeostasis in light condition. Plant J 40:333–343CrossRefPubMedGoogle Scholar