SYNTHETIC DNA PROBES TO IDENTIFY MEMBERS OF THE ANOPHELES-GAMBIAE COMPLEX AND TO DISTINGUISH THE 2 MAJOR VECTORS OF MALARIA WITHIN THE COMPLEX, ANOPHELES-GAMBIAE SS AND ANOPHELES-ARABIENSIS

Two cloned DNA sequences, lambda C10 and lambda G12, have been isolate d from a female Anopheles gambiae sensu stricto genomic DNA library in lambda EMBL4. The lambda C10 clone hybridized with equal intensity to all five of the six species in the An, gambiae Giles complex tested a nd was therefore suitable for use as a complex-specific clone. The lam bda G12 clone was selected for its ability to distinguish the two majo r vectors of malaria within the complex, An. gambiae s.s. and An. arab iensis. Use of libraries consisting of only female DNA prevented the i solation of male-specific sequences. Southern blot analysis of the clo ned DNA permitted the development of smaller Alu I subclones suitable for sequencing that still retained the original specificities and sens itivities of lambda C10 and lambda G12. Each clone was found to posses s a series of repeated sequences in direct tandem array of 92-94 and 6 8 bases, respectively. A comparison of a number of copies of each of t he repetitive sequences within the Alu I subclones enabled the definit ion of consensus sequences for the repetitive elements in lambda C10 a nd lambda G12. Based on these consensus sequences, two oligonucleotide s of 21 and 23 bases designated pAngsl and pAngss were derived from la mbda C10 and lambda G12, respectively. When tested as probes against D NA dot-blots and squash-blots of mosquito specimens, each oligonucleot ide retained the same species specificity as the original clones from which they were derived. The nonradioactive, alkaline phosphatase-labe led pAngsl was able to detect as little as 1 ng of target genomic DNA by chemiluminescent detection in a 5-hr autoradiographic exposure. The pAngss probe could detect 5-10 ng of genomic DNA in similar assays. T he new probes exhibit great potential for use in An. gambiae complex s pecies identification because they provide both a means of distinguish ing the two major vectors of malaria within the complex and of assessi ng the quality of squashed mosquito samples by providing a means of st andardizing hybridization results.