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DNA Laboratories

General information

A correct species identification is crucial for policy concern issues such as: endangered species protected under the CITES treaty, Invasive Alien Species, agricultural pest species, human and veterinary disease organisms and their vectors, organisms of the human food chain, forensically interesting species, species with economic impacts, etc.

When a morphological identification is not possible to enforce the respective regulations, DNA barcoding can often offer a solution. This technique uses short DNA sequences as barcodes by comparing and matching the unidentified query sequences to known, identified sequences in reference libraries.

DNA sequences, from either unknown samples that must be identified or voucher material for reference databases, are produced in a laboratory environment. The laboratories used by BopCo are located at the Royal Museum for Central Africa (RMCA) and the Royal Belgian Institute for Natural Sciences (RBINS). Here DNA is extracted and purified from tissues or processed materials and a specific region of the genetic material is amplified. This process of DNA amplification is called polymerase chain reaction (PCR) and most often the mitochondrial cytochrome c oxidase 1 (COI) gene is the region of choice for animals, as defined by the International Barcode of Life initiative. Subsequently, the PCR products are purified and prepared for sequencing in-house or by an external sequencing facility. The generated sequences are then analyzed and the species is identified using online reference databases. Where applicable, policy advice can be given specific to the issue at hand.

 

Infrastructure / Installation

Currently (September 2017) BopCo has access to five laboratories which are partly equipped with BopCo instruments. Both in the RBINS and RMCA buildings the lab facilities are separated over two rooms: a pre-PCR and post-PCR environment in order to prevent cross-contamination.

In the pre-PCR lab caution is required to avoid contamination with DNA from the environment, especially when aliquoting reagents or when working on sensitive materials. In this environment the sterilization of all consumables is carried out using an autoclave and a specially allocated oven to fully dry the plastics before use. The procedures carried out in the pre-PCR lab include: (i) taking tissue samples from specimens, (ii) extracting DNA using either standardized kits or customized protocols, (iii) measuring the DNA concentration of the extracts, (iv) preparing the PCR mixtures, and (v) storing the DNA samples in assigned -20 °C freezers for later use.

When working with very sensitive or old samples which contain only little or potentially degraded DNA, a specially equipped ancient DNA lab is available at the RBINS. This specialized room is supplied with UV light and positive pressure airflow to avoid contamination with DNA from the environment.

    

Left: Typical pre-PCR workbench - Right: pre-PCR equipment left to right: centrifuge, vortex mixer, three shaking heat blocks and a water bath. (Photographs ©BopCo)

 

In the post-PCR room the prepared DNA extracts are subjected to PCR amplification of the selected DNA fragment. This process requires thermocyclers; machines that can ramp and hold temperature precisely for short periods of time. To confirm that the PCR reaction has worked the amplification results are visualized using agarose gel electrophoresis with UV transillumination. After this inspection, the successful PCR products are purified, clearing away leftover PCR reagents, and the purified PCR product is prepared for sequencing.

    

Left: Thermal cycler with on-screen cycling protocol - Right: gel electrophoresis tank and power supply - Insert: example of visualization of PCR results. (Photographs ©BopCo)

 

An ABI 3130XL Sequencer is available for use by the BopCo team in the RBINS laboratories. Sequencer output files can be analyzed and compared on specialized programs.

The RBINS ABI 3130XL Sequencer in use and inserted an example output file in Geneious software. (Photographs ©BopCo)

 

Additionally, BopCo has storage facilities for the preservation of both tissue samples and DNA-extracts. The tissue and specimen samples are stored in -80 °C freezers, while the DNA extracts are preserved using the GenTegra drying technology for the long-term storage of DNA at room temperature and kept in Abgene 2D Barcode tubes in a GeneVault.

Illustration of Abgene 2D barcode tubes and storage boxes with VisionMate scanner (©ThermoFisher Scientific)

 

The fact that BopCo is jointly run by RBINS and RMCA means that it has easy access to the taxonomic expertise available at both research institutes, as well as to the large curated collections of specimens.

 

Applications / Data flow

Instead of developing a separate web portal, the barcode sequences produced by BopCo, as well as the associated/accompanying sample information is made available via the Barcode Of Life Data Systems (BOLD Systems) database platform, where the sequences are publicly available for consultation and analysis.

To learn more about BopCo’s activities and research projects, visit http://bopco.myspecies.info/.