1921
Volume 50, Issue 3
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645
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Abstract

Abstract

Two cloned DNA sequences, λC10 and λG12, have been isolated from a female sensu stricto genomic DNA library in λEMBL4. The λC10 clone hybridized with equal intensity to all five of the six species in the Giles complex tested and was therefore suitable for use as a complex-specific clone. The λG12 clone was selected for its ability to distinguish the two major vectors of malaria within the complex, s.s. and . Use of libraries consisting of only female DNA prevented the isolation of male-specific sequences. Southern blot analysis of the cloned DNA permitted the development of smaller I subclones suitable for sequencing that still retained the original specificities and sensitivities of λC10 and λG12. Each clone was found to possess a series of repeated sequences in direct tandem array of 92–94 and 68 bases, respectively. A comparison of a number of copies of each of the repetitive sequences within the I subclones enabled the definition of consensus sequences for the repetitive elements in λC10 and λG12. Based on these consensus sequences, two oligonucleotides of 21 and 23 bases designated pAngsl and pAngss were derived from λC10 and λG12, respectively. When tested as probes against DNA dot-blots and squash-blots of mosquito specimens, each oligonucleotide retained the same species specificity as the original clones from which they were derived. The nonradioactive, alkaline phosphatase-labeled 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. The new probes exhibit great potential for use in complex species identification because they provide both a means of distinguishing the two major vectors of malaria within the complex and of assessing the quality of squashed mosquito samples by providing a means of standardizing hybridization results.

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/content/journals/10.4269/ajtmh.1994.50.312
1994-03-01
2017-11-21
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