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Minerva Biotecnologica 2018 March;30(1):7-13

DOI: 10.23736/S1120-4826.17.02294-7


language: English

Primer Spanner: a web-based platform to design PCR primers for high efficient site-directed mutagenesis and DNA assembling

Xianhui HOU 1, 2, Zhiyong PEI 1, 2, Xiaoxing WEI 1, 3

1 State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Xining, China; 2 Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing, China; 3 Basic Medical Science Research Center, Medical College of Qinghai University, Xining, China

BACKGROUND: PCR-based strategy has economical and efficient application in site-directed mutagenesis (SDM) and DNA assembling such as PCR-based vector construction. In these processes, primers are extremely important as they determine the amplification efficiency of the target DNA fragments, and also influence circularization of the new plasmids, so they determine the success rate. There are several types of primer designing software for regular gene cloning PCR, however, they are not suitable to design primers for SDM or vector construction. Different evaluation criteria are required for such chimeric primer design, in particular for high-throughput purposes.
METHODS: Each primer comprises the 3’-complete matching part (CM) and 5’-overlapping part (OL) for SDM and DNA assembling. Tm of each part should be calculated separately, due to different roles they play in PCR. For SDM, only the input sequences and mutation codes are needed. The mutation code determines the position and type of mutagenesis including substitution, insertion and deletion. For DNA assembling e.g. vector construction, only two joint sequences (insertion fragment and target plasmid) and junction codes are needed to input. The PCR products can be transformed to E. coli competent cells with or without DpnI digestion according to the existence of template plasmid contamination.
RESULTS: An easy-to-use platform was developed to generate primers automatically according to the input sequences and mutation codes. Primers were evaluated and optimized with Tm, GC%, secondary structure and length. Near 100% success rate was achieved in our validation assay for all kinds of mutagenesis and 16 substitutions were introduced at one time using 4 pairs of primers generated by this program.
CONCLUSIONS: “Primer Spanner” (PS) is a special tool for chimeric primer designing, and is applicable for SDM including single-site or multi-site substitution, insertion and deletion with high successful rate. It can also be used in DNA assembling primer design for both linear DNA and ligase-free vector construction. The software is freely accessible at: http://PS.biocloud.org.cn/.

KEY WORDS: Polymerase chain reaction - Mutagenesis, site-directed - Genetic vectors

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