Note: An Improved COII Primer Specific for Lepidoptera

Abstract

DNA fragments of the mitochondrion have been considered as powerful molecular markers for phylogenetic analysis of animals. The general strategy is amplification of a certain fragment through forward and reverse primers, and subsequent sequencing. The primers are usually designed in the conserved regions at the two ends of a particular gene in order to use the primers in a variety of organisms, i.e., universal or conserve primers. Simon et al. (1994) reported many universal primers for all fragments of insect mitochondria. These primers have been extensively used in phylogenetic analysis of many insect taxa. Among the encoding regions, the cytochrome oxidase subunit II (COII) fragment is analyzed and sequenced in many species of insect. To amplify the whole COII region, TL2-J-3037 (abbr. TL2 in this paper) and TK-N-3785 are considered as a pair of universal primers for insects (Simon et al., 1994). TL2 is located in the Leu-tRNA region at the 5′-end of COII, and TK-N-3785 is located in the Lys-tRNA region at the 3′-end of the COII gene. This primer pair has been used successfully in many insect taxa, including Diptera, Coleoptera, Orthoptera, and Lepidoptera (Liu and Beckenbach, 1992). However, when we used this primer pair to amplify COII in Polyura eudamippus (Lepidoptera: Nymphalidae), we found that some of the samples could not be amplified, and it was not due to failure of the template DNA as some of these samples have been amplified in another amplification. After we improved the PCR conditions, such as increasing Mg2+ concentration, increasing the concentration of template DNA and primers, and adding BSA, the number of amplified samples did not increase. Even if we decreased annealing temperature to 45◦C, there was no improvement. A similar situation also occurred in a study on Ostrinia furnacalis (Lepidoptera: Pyralidae) in our lab. Kim et al. (1999) also found that this primer pair did not work on O. zealis.

DOI: 10.1023/A:1020926613349

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Cite this paper

@article{Wang2004NoteAI, title={Note: An Improved COII Primer Specific for Lepidoptera}, author={Rongjiang Wang and Yu Long}, journal={Biochemical Genetics}, year={2004}, volume={41}, pages={57-60} }