RESUMO
This proof-of-principle study describes the development of a rapid and easy-to-use DNA microarray assay for the authentication of giant tiger prawns and whiteleg shrimp. Following DNA extraction and conventional end-point PCR of a 16S rDNA segment, the PCR products are hybridised to species-specific oligonucleotide probes on DNA microarrays located at the bottom of centrifuge tubes (ArrayTubes) and the resulting signal patterns are compared to those of reference specimens. A total of 21 species-specific probes were designed and signal patterns were recorded for 47 crustacean specimens belonging to 16 species of seven families. A hierarchical clustering of the signal patterns demonstrated the specificity of the DNA microarray for the two target species. The DNA microarray can easily be expanded to other important crustaceans. As the complete assay can be performed within half a day and does not require taxonomic expertise, it represents a rapid and simple alternative to tedious DNA barcoding and could be used by crustacean trading companies as well as food control authorities for authentication of crustacean commodities.
Assuntos
DNA/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Penaeidae/genética , Animais , Astacoidea/genética , RNA/genética , RNA Ribossômico 16S/genética , Especificidade da EspécieRESUMO
Cluster of differentiation 24 (CD24) is a cell surface glycoprotein, which is largely present on hematopoietic cells and many types of solid tumor cells. CD24 is known to be involved in a wide range of downstream signaling pathways and neural development, yet the underlying mechanisms are poorly understood. Moreover, its production correlates with poor cancer prognosis, and targeting of CD24 with different antibodies has been shown to inhibit disease progression. Nucleic acid aptamers are oligonucleotides that are selected from random DNA or RNA libraries for high affinity and specific binding to a certain target. Thus, they can be used as an alternative to antibodies. To gain an insight on CD24 role and its interaction partners, we performed several SELEX (systematic evolution of ligands by exponential enrichment) experiments to select CD24-specfiic DNA aptamers. We found that the cell-SELEX approach was the most useful and that using HT-29 cell line presenting CD24 along with CD24 knockdown HT-29 cells has selected six aptamers. For the selected aptamers, we determined dissociation constants in the nanomolar range (18-709 nM) using flow cytometry. These aptamers can be applied as diagnostic tools to track cancer progression and bear a potential for therapeutic use for inhibiting signaling pathways that promote the metastatic process.