JRA4: Plants/fungi optimised DNA Extraction Techniques

Plant/fungal sample destruction in herbarium collections is not so much the issue but rather more intrinsic factors effecting DNA extraction success. The key technical issue to overcome is the masking effects of secondary compounds plus the unique presence of cell-walls that present a major challenge for effective DNA extraction. Impressive results from herbarium specimens up to 100 years old have been reported, but oxidative damage to their DNA may still prevent extraction of correct sequences. JRA4 aims to assess this kind of damage by applying the new 454 sequencing technology.

Aims and objectives

JRA4 will develop new protocols for Users in the systematic community to enable them to carry out the efficient mining of herbarium collections for DNA utilising state-of-the-art DNA technology, and thus enhancing the value of specimen collections and contribute to DNA banks.

Among the most valuable herbarium collections are rare species and type specimens, many of which are quite old and none of which can easily be re-collected. It is therefore crucial to develop protocols making these specimens in particular accessible for DNA extraction and sequencing. Accordingly, our emphasis will be on optimising protocols for older material, >70 years. Another focus will be on the optimisation of DNA extraction from (stem) wood.

The outcome of JRA4 will be to substantially increase the value of existing global herbarium collections. It will facilitate the more rapid population of DNA banks with scientifically significant and vouchered, taxonomically-verified DNA. Providing the means for efficient DNA liberation from herbarium collections will increase their accessibility to Users resulting in wider application for the research community, such as DNA-based systematic, ecological or population genetics studies.
There are 2 main objectives of JRA4:

  • Identify the main obstacles to successful DNA extraction/amplification of herbarium specimens based on user experiences
  • Develop optimal protocols for the extraction of DNA from old, alcohol-preserved, and secondary metabolite-rich herbarium specimens of a wide range of plants and fungal clades, with a view to enabling subsequent large-scale and high-throughput automation.

Description of work

Oxidative DNA damage in herbarium specimens & 454 sequencing

Make family × age × preservation factor comparison for assessing (and interpreting) oxidative damage in old herbarium specimens of both (angiosperm) plants and fungi by exploring both the analytical power offered by 454 sequencing and by consultation with lab managers and users from the botanical/mycological community.

Extensive within-run labelling in this technology enables direct and relative assessment of i) oxidative damage in multiple samples of the same species collected at different times and with different methods, ii) relative preservation of different tissues from the same organism, and iii) different preservation types within the same tissue.

The use of PrediCtoR software (see JRA1) for initial assessment of herbarium specimens will also explored.

Secondary metabolites

Identification and testing of secondary metabolite-specific solutions to purifying DNA bound to these compounds will take place. This will focus mostly on plants but will include selected fungi.

DNA extraction from alcohol-preserved herbarium specimens of plants

For old alcohol-preserved material existing CTAB protocols will either be improved or new ones developed. Phased re-hydration of dried and preserved materials will be explored.

Testing the protocols

Protocols developed in the other tasks will be subject to large scale tests in selected institutions with collections reflecting different storage conditions and durations, express success of yields and quality of results relative to that using shared positive controls (panel of standard specimens). This will lead to the recommendation for future fungal and plant specimen preservation.

Progress reports

Work started in Sept 2009. Nothing to report yet.

Outcomes and deliverables

Events

There are no planned events at this stage.

How can I get involved

The protocols will need to be tested. This is expected to take place in September 2011. Please check back for more details.

For more information contact synthesys@nhm.ac.uk.