Occurrence of an unusual polymerase chain reaction product during identification of Anopheles gambiae sibling species (Diptera: Culicidae)

Abstract

 Using the polymerase chain reaction (PCR) for species differentiation within the Anopheles gambiae complex, we examined several hundred Cameroonian mosquito specimens. Applying an approved routine protocol for DNA extraction and PCR conditions, apart from the indubitable identification of most of the specimens, we came across ten PCR products that did not correspond in length to any of the hitherto known amplification products of the sibling species. Sequencing experiments showed the ten products to be of identical length (117 bp) and nucleotide sequence. The total sequence of the novel product is included in the PCR product specific for A. melas, which is known to occur in the same collection area as the ten unidentifiable mosquito specimens. On alignment of the novel PCR product sequence and the A. melas one starting at the 3"-end primer annealing site, the last 20 nucleotides of the novel product, reflecting the sequence of the 2^nd PCR primer, showed only 60% homology with the then-corresponding A. melas DNA site. Explanations for the occurrence of the unusual PCR products are given and discussed.