Universal primers for the amplification and sequence analysis of actin-1 from diverse mosquito species

We report the development of universal primers for the reverse-transcription polymerase chain reaction (RT-PCR) amplification and nucleotide sequence analysis of actin cDNAs from taxonomically diverse mosquito species. Primers specific to conserved regions of the invertebrate actin-1 gene were desig...

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Bibliographic Details
Published inJournal of the American Mosquito Control Association Vol. 26; no. 2; p. 214
Main Authors Staley, Molly, Dorman, Karin S, Bartholomay, Lyric C, Fernández-Salas, Ildefonso, Farfan-Ale, Jose A, Loroño-Pino, Maria A, Garcia-Rejon, Julian E, Ibarra-Juarez, Luis, Blitvich, Bradley J
Format Journal Article
LanguageEnglish
Published United States 01.06.2010
Subjects
Actins - chemistry
Actins - genetics
Actins - metabolism
Animals
Base Sequence
Culicidae - classification
Culicidae - genetics
Culicidae - metabolism
DNA Primers
Phylogeny
Reverse Transcriptase Polymerase Chain Reaction
sequence
actin
reverse transcription-polymerase chain reaction
universal primers
Mosquito
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Summary:We report the development of universal primers for the reverse-transcription polymerase chain reaction (RT-PCR) amplification and nucleotide sequence analysis of actin cDNAs from taxonomically diverse mosquito species. Primers specific to conserved regions of the invertebrate actin-1 gene were designed after actin cDNA sequences of Anopheles gambiae, Bombyx mori, Drosophila melanogaster, and Caenorhabditis elegans. The efficacy of these primers was determined by RT-PCR with the use of total RNA from mosquitoes belonging to 30 species and 8 genera (Aedes, Anopheles, Culex, Deinocerites, Mansonia, Psorophora, Toxorhynchites, and Wyeomyia). The RT-PCR products were sequenced, and sequence data were used to design additional primers. One primer pair, denoted as Act-2F (5'-ATGGTCGGYATGGGNCAGAAGGACTC-3') and Act-8R (5'-GATTCCATACCCAGGAAGGADGG-3'), successfully amplified an RT-PCR product of the expected size (683-nt) in all mosquito spp. tested. We propose that this primer pair can be used as an internal control to test the quality of RNA from mosquitoes collected in vector surveillance studies. These primers can also be used in molecular experiments in which the detection, amplification or silencing of a ubiquitously expressed mosquito housekeeping gene is necessary. Sequence and phylogenetic data are also presented in this report.
ISSN:8756-971X
DOI:10.2987/09-5968.1