Are “universal” DNA primers really universal?

Pranay Sharma*, Tsuyoshi Kobayashi

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

17 Citations (Scopus)


“Universal” DNA primers LCO 1490 and HCO 2198 were originally designed from three coding and six anticoding strands by comparing highly conserved regions of mitochondrial cytochrome c oxidase subunit I (COI) genes across 15 taxa. These primers have been successful in amplifying a 710-bp fragment of highly conserved regions of the COI gene formore than 80 invertebrate species from 11 phyla. In the present study, 130,843 variations were reviewed in the primer region of mitochondrial molecular markers by comparing 725 COI sequences fromthe kingdom Animalia. It was found that, for 177 invertebrate species, the forward primer (LCO 1490) showed only four conserved regions, compared to 12 in the original study. For ascidians, fungi and vertebrates, it showed approximately 50% conserved regions, dropping to one conserved region for echinoderms. However, the reverse primer (HCO 2198) was highly conserved across 725 COI primer sequences. A similar pattern was observed in amino acid distributions. There was a significant difference in the means of base pair differences from the level of family, genus and species for LCO 1490 [analysis of variance (ANOVA), F6,188=8.193, P<0.001] and at the level of genus and species for HCO 2198 (ANOVA, F6,77=2.538, P<0.027). We conclude that, at different taxonomic levels, it is possible to design forward primers from reference sequences belonging to the level of order (maximum 5 bp differences), family (maximum 6 bp differences) or genus (maximum 1 bp difference). Reverse primers can be designed from the level of family (maximum 5 bp differences) or genus (maximum 2 bp differences).

Original languageEnglish
Pages (from-to)485-496
Number of pages12
JournalJournal of Applied Genetics
Issue number4
Publication statusPublished - Nov 2014
Externally publishedYes


  • Folmer primers
  • MtDNA
  • NCBI
  • Universal


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