JRA3 will make the first attempt to develop a system for the use of very small samples (microsamples) in ancient DNA extraction from museum bone specimens. It will investigate the full range of issues arising from the use of microsamples, including the practicalities of sampling, extraction, heterogeneous DNA distribution and increased contamination risk. The success of JRA3 will open up the majority of the bone specimens that were previously not available for sampling, as they are much too valuable to permit user access. This includes type specimens, bone artefacts and very small specimens.
A reliable and consistent DNA extraction method is fundamental to any attempt by users of museum materials to undertake advanced genetic studies. Most of the available DNA extraction methods used in palaeogenetics are based on classical lab protocols, such as solvent extraction, and include steps such as ethanol precipitation and sedimentation by centrifugation that typically result in a considerable reduction of DNA yield. Most studies employ samples of several hundred mg or more in mass. Taking into account the necessity of reproduction of the extraction experiments, and material lost during the sampling process, typically more than half a gram of sample is required. This amount is far too much for small and rare samples of cultural and scientific importance. A significant barrier to the development of better DNA extraction methods, which would enable the routine and reliable recovery of genetic material from extremely small sample volumes, is that the mechanism by which DNA is preserved in bone over long periods remains as yet unclear.
Although a range of studies of DNA extraction methods for archival bone material have been published, there has been no systematic attempt to develop a system for using micro-samples (less than 0.005g mass). While the use of completely non-invasive techniques has been discussed, this is only practical for samples which are small enough to be completely immersed in extraction buffer. Further, the microstructural effects of such extraction are unknown, but are thought likely to significantly reduce the long-term mechanical properties of the material. Micro-sampling by high speed drilling is a more attractive option to maintain the aesthetic, morphometric and mechanical properties of small and culturally valuable specimens. The overall objectives of JRA3 are to:
- Generate baseline data on the use of minimally-destructive bone sampling techniques for application in genetic studies of museum archive-held material
- Improve our understanding of the location and stabilisation of DNA in bone, to develop optimal methods for DNA recovery, pre-screening and decay modelling
- Provide Users with an improved protocol for the recovery of pre-amplified DNA from extremely small sample volumes
Experimental assessment of the minimal sample size for which it is realistic and feasible to conduct DNA successful extraction
The extent to which these extremely small volumes of bone, can be recovered “in the field”, cleaned for removal of contaminant DNA, museum consolidants or preservatives, and processed for DNA extraction is unknown. JRA3 will work with a range of material from museum specimens and with previously characterised material, in order to develop a best-practice method for minimally-invasive sampling.
Protocols for the extraction of highly degraded DNA from human or hominid specimens
The risk of contamination by archaeologists, curators, anthropologists, or simply the museum environment must be taken into account. There are already several protocols that describe the appropriate anti-contamination measurements, but still a completely human-contamination-free ancient DNA extraction protocol has not been developed. Therefore, JRA3 will work on the improvement of decontamination protocols for human samples.
A completely new light on this contamination issue is thrown by the possibility of applying 454 sequencing on ancient DNA. This new technique is expected to become a standard in palaeogenetics within the life of SYNTHESYS. Though it does not change the number and rate of contaminating molecules, in real terms it will make possible a far more accurate differentiation between authentic and contaminating molecule by looking at mass data generated from an ancient DNA extract or its PCR products. The decontamination procedures that we plan to develop by 36m will be used directly in the subsequent application of 454 sequencing.
Evaluation of the extent to which DNA in bone is heterogeneously distributed.
Extract DNA from multiple micro-samples of individual bones, in order to test the extent to which DNA in bone is heterogeneously distributed. The degree to which this is the case, i.e. that a single element contains DNA-rich and DNA-poor regions, is an important element in determining the utility of the micro-sampling approach and will feed back into the analysis strategy used by JRA2, and the results from JRA1.
Bone is a well described tissue, and the potential sources of DNA in bone are known. Post-mortem, it seems most likely that it is bound to the hydroxyapatite matrix, the mineral phase of the bone. It has been demonstrated that aggregations of hydroxyapatite crystals are present in most bone types, and that there appears to be organic material trapped within these crystalline aggregates. It has been proposed that these aggregate organic cores contain a useful source of uncontaminated DNA This prediction so far remains largely untested and new knowledge about this is badly needed. In order to generate this new knowledge we propose to:
- Mill several bone samples and sort the resulting powder using a combination of sieving and sedimentation approaches
- Sequentially bleach bone fractions to remove surface DNA
- Extract the fractions and quantify the DNA in each using quantitative PCR
In this way, it should be possible to develop a profile of DNA preservation across different size fractions of the bone powder, and by reference to the known histological properties of bone, establish the micromorphological features of bone, which preserve DNA.
Development of new assay methods for the optimal recovery of DNA from bone micro-samples
Currently applied methods for DNA extraction generally rely on partially dissolving the mineral matrix and then recovering DNA from the soluble or insoluble fractions using either solvent extraction and subsequent aqueous phase concentration, or DNA-binding via chaotropic salt. Microsamples are likely to completely dissolve into aqueous solution, and may therefore require alterations to these standard protocols in order to result in sufficient DNA recovery, without co-purification of compounds inhibitory to downstream applications. JRA3 will screen a suite of approaches to establish the optimal procedure for efficient and reliable recovery of DNA, using material sampled in the first task.
Validation of the microsampling protocol will be carried out on a set of material identified by application of the protocols developed in JRA1 and JRA2.
The central direction of the work of JRA3 has been to develop a system for the use of very small samples (microsamples) in
ancient DNA extraction from museum bone specimens.
Protocol for contamination free DNA extraction from human bone for subsequent 454 sequencing reported (Deliverable 6.2 of the SYNTHESYS2 project; incorporated in the JRA2 Protocols report)
Validated, optimal DNA extraction protocol for microsamples (Deliverable 6.4)
Ancient DNA and evolution conference. 14th October 2009 – Natural History Museum, London. Click here for more details.
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