PCR-based methods for identification of species of the Anopheles minimus group: allele-specific amplification and single-strand conformation polymorphism.
We report two polymerase chain reaction (PCR)-based methods for distinguishing morphologically similar species based on amplification of a variable region of the 28S gene of ribosomal DNA. The four species we investigated are mosquitoes of the Anopheles minimus group: An. aconitus, An. varuna and An. minimus species A and C. The formally named species are vectors of human malaria parasites in south-east Asia but are difficult to distinguish with certainty on the basis of morphology. Allele-specific amplification was used to differentiate An. minimus A from An. minimus C. This technique has been widely used for the diagnosis of species. Single-strand conformation polymorphisms (SSCPs) were used to separate all four species. This technique, which has seldom been used for species identification, has many advantages: it does not require sequence information beyond that needed for amplification; it is ideally suited for the detection of heterozygotes; it utilizes more of the information in the PCR product than allele-specific amplification; it distinguishes all four species considered here and could easily be extended to other species; previously unknown intraspecific variation and additional species are likely to be detected. Thus, SSCPs provide valuable population genetic information which allele-specific amplification does not.
Sharpe
R G
RG
School of Biology, The University of Leeds, UK. genrgs@leeds.ac.uk
Hims
M M
MM
Harbach
R E
RE
Butlin
R K
RK
D000483:Alleles; D000818:Animals; D000852:Anopheles / Q000145:classification / Q000235:genetics*; D002417:Cattle; D004275:DNA, Ribosomal / Q000032:analysis; D005260:Female; D005784:Gene Amplification; D017344:Genes, Insect; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D012339:RNA, Ribosomal, 28S
D004275:DNA, Ribosomal; D012339:RNA, Ribosomal, 28S