JRA5 works alongside to JRA4, for the development of a new extraction protocol for groups of invertebrates. It aims to replace time-consuming single sample extractions by a high-throughput DNA isolation procedure, plus improve the yield from old samples, which is poor with existing protocols.
Invertebrates make up an important part of all major European museum collections and taxa that contain a significant amount of muco-polysaccharides present a problem to molecular research. Both DNA and muco-polysaccharides are extracted together if current, standard methods are applied. This severely limits the downstream application of the most common standard genetic analysis method (PCR).
It is not yet known how the muco-polysaccharide problem is affected by the prolonged storage (more than ten years) of samples in ethanol, but the problem likely persists. Alternative methods such as magnetic bead technology have recently been developed, but their applicability to this specific problem has not yet been tested.
The outcome of JRA5 will be to substantially increase the value of existing museum collections by making them more readily accessible for future DNA research.
There are 2 main objectives of JRA5:
- Develop and test safer and more effective DNA isolation procedure from muco-polysaccharide-rich tissue, focussing on museum specimens of varying age (5-150 years)
- Setup a Web database as an efficient tool to disseminate protocols and experience of DNA extraction from non-entomological invertebrates reports among Users
Development of alternatives to CTAB for the separation of DNA from polysaccharides
The present CTAB protocol is not adaptable to high-throughput methods due to the requisite separation of two liquid phases. An enzyme-based method using amylases to remove the muco-polysaccharides before DNA purification seems most appropriate, both in terms of reliability and cost-effectiveness.
In parallel, magnetic bead technologies will be tested for their effectiveness in getting clean DNA from muco-polysaccharide-rich tissue and modified, if necessary. Depending on the success of both the amylase-based procedure and the magnetic bead method, either one of the methods will be selected or they may be combined.
Testing of replacement components for β-mercaptoethanol
The efficiency of tissue break-up and DNA recovery using alternatives (e.g. DTT, TCEP-HCl) will be compared. Prior to testing alternatives to CTAB the need for using a disulfide bond breaking reagent will be assessed.
Optimisation of the selected method f for high-throughput procedures.
The results of the previous two tasks will be analysed and the procedures optimized. This will also include methods of initial mechanical tissue break-up, which consumes a relatively large proportion of time in the DNA extraction process.
Development and implementation of large-scale tests of the new protocols.
A jointly supported database will be used to allow the monitoring of testing progress. If significant deviations from the success rate experienced during protocol development become apparent during this phase, attempts will be made to adjust the protocol to either collection-specific or taxon -specific problems. High failure rates that can be traced to certain storage or sampling handling conditions can be used to issue recommendations both in published papers and particularly on the internet. Access Users and other visitors will be invited to submit their findings to the database.
Development of a Web database for the dissemination of results among Users.
A database will be established to facilitate the monitoring of success rates particularly during the large-scale testing and the dissemination of emerging issues. It will subsequently provide the improved protocol or set of protocols to the scientific community including, Users given access to the SYNTHESYS institutions and other collections-holding institutions globally.
Presentation of alternative method for DNA extraction from mucopolysaccharide rich tissue (Deliverable 8.1 of the SYNTHESYS2 project).
Report on tested replacement component for β-mercaptoethanol (Deliverable 8.2).
Present protocol adapted to high-throughput methods (96-well format) (Deliverable 8.3).
Complete extensive parallel testing in several institutions of safe high throughput method for DNA extraction from muco-polysaccharide rich tissue with notes on general applicability (Deliverable 8.4).
Work started in Sept 2009. Nothing to report yet.
The protocols will need to be tested. This is expected to take place in September 2011.
Entering of data onto the website when it is developed.
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