Isolating cells from non-sperm cellular mixtures using the PALM® microlaser micro dissection system
Introduction
Forensic scientists are sometimes confronted with cellular material originating from an individual, often in trace quantities, that is mixed with cellular material from a different person. DNA profiling of such cellular mixtures will often either generate mixed DNA profiles that require complex statistical calculations [1] or a profile that only represents the major contributor, when the need is to obtain DNA information on the minor contributor. At present, the forensic community has no reliable technique for separating trace amounts of somatic (non-sperm) cells from cellular mixtures. In addition to generating an informative, single-source DNA profile, knowledge of the cell type contributing the DNA profile can be relevant [2].
Microlaser dissection systems, combining microscope and laser cutting and isolation beam technology, permit the identification and isolation of tissue sections and single cells, as well as sub-cellular components [3]. Microlaser systems are used by the forensic community to isolate sperm from vaginal cell mixtures [4], [5], [6], the lower follicle from telogen hair [7], fetal from maternal tissue [8], [9], and male cells from cellular mixtures [10], [11]. Previously we have reported use of the Positioning Ablation Laser MicroBeam® (PALM®) MicroLaser system (P.A.L.M. Microlaser Technologies AG, Bernried, Germany) to identify and isolate cells from environmental debris [12]. Here we investigate the ability of the PALM® microlaser to identify and isolate trace amounts of cells from somatic cellular mixtures (blood and saliva), and compared its efficiency in generating single-source DNA profiles with current profiling protocols.
Section snippets
Sample preparation
Immediately following collection, blood (donor A) and saliva (donor B) were mixed, together with TE buffer (10 mM Tris–HCl, 0.1 mM EDTA) to inhibit blood coagulation. The following samples were prepared: Mixture 1 (25% blood, 5% saliva, 70% TE), Mixture 2 (25% blood, 10% saliva, 65% TE), Mixture 3 (25% blood, 50% saliva, 25% TE), Mixture 4 (5% blood, 50% saliva, 45% TE) and Mixture 5 (2.5% blood, 50% saliva, 47.5% TE); the percentages represent their volume within a given mixture. Each mixture (20
DNA profiling of mixtures
All mixtures, except Mixture 1, yielded DNA profiles that contained alleles from both donors (Table 1). Mixture 1 (25% blood and 5% saliva) only gave the blood donor's DNA profile. Mixture 2 (25% blood and 10% saliva) yielded all unique alleles of the blood donor, along with four (of 14) unique alleles from the saliva donor, whilst Mixtures 3 (25% blood and 50% saliva), 4 (5% blood and 50% saliva) and 5 (2.5% blood and 50% saliva) contained all the unique alleles of blood and saliva donors.
Discussion
We have shown how the PALM® microlaser improves the chances of obtaining informative, single-source DNA profiles from somatic cellular mixtures. Under present protocols such mixtures would either generate mixed DNA profiles or reveal only the major cell component. Because of the unknown parameters surrounding transfer of trace cellular material to a crime scene, the evidential significance of a DNA profile match between a crime scene and suspect may be limited. In certain case scenarios
Acknowledgements
This work was supported by an Australian Research Council Linkage grant and the Victorian Police Forensic Services Department.
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