Microcoding: the second step in DNA barcoding

Abstract

After the process of DNA barcoding has become well advanced in a group of organisms, as it has in the economically important fungi, the question then arises as to whether shorter and literally more barcode-like DNA segments should be utilized to facilitate rapid identification and, where applicable, detection. Through appropriate software analysis of typical full-length barcodes (generally over 500 base pairs long), uniquely distinctive oligonucleotide 'microcodes' of less than 25 bp can be found that allow rapid identification of circa 100-200 species on various array-like platforms. Microarrays can in principle fulfill the function of microcode-based species identification but, because of their high cost and low level of reusability, they tend to be less cost-effective. Two alternative platforms in current use in fungal identification are reusable nylon-based macroarrays and the Luminex system of specific, colour-coded DNA detection beads analysed by means of a flow cytometer. When the most efficient means of rapid barcode-based species identification is sought, a choice can be made either for one of these methodologies or for basic high-throughput sequencing, depending on the strategic outlook of the investigator and on current costs. Arrays and functionally similar platforms may have a particular advantage when a biologically complex material such as soil or a human respiratory secretion sample is analysed to give a census of relevant species present.

Figure 1

Annealing of labelled amplicon with a specific oligonucleotide that is linked to a solid surface via an amino linker.

Figure 2

Macroarray exposed to Pythium- and Phytophthora-specific nuclear ribosomal internal transcribed spacer-region amplicons obtained from a DNA sample extracted from field-grown soybean roots (T. Barasubiye, Agriculture and Agri-Food Canada, unpublished). Dark spots appear where specific oligonucleotides have captured labelled amplicons corresponding to various species present on the roots. The array is covered by a mask indicating the identity of the various spots seen. ‘X’ symbols are placed over known cross-reacting oligonucleotides.