Isolating cells from non-sperm cellular mixtures using the PALM® microlaser micro dissection system

doi:10.1016/j.forsciint.2007.01.031

Forensic Science International

Volume 173, Issues 2–3, 20 December 2007, Pages 93-96

https://doi.org/10.1016/j.forsciint.2007.01.031 Get rights and content

Abstract

Use of the Positioning Ablation Laser MicroBeam^® (PALM^®) microlaser system to isolate specific cellular components from somatic cellular mixtures (blood and saliva) prior to DNA extraction and typing is compared with routine DNA extraction and typing of the same mixture samples. Mixtures of blood and saliva at differing ratios generated complex DNA profiles that included allele peaks originating from each of the donors, or just those of the major contributor. Isolation of cells with the PALM^® microlaser prior to DNA extraction allowed informative, single-source, DNA profiles to be generated from a known cell type/origin.

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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Use of fluorescence in situ hybridisation and laser microdissection to isolate male non-sperm cells in cases of sexual assault
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Laser microdissection and pressure catapulting with PALM^® to assist typing of target DNA in dirt samples
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A comparison of the characteristics of profiles produced with the AMPFlSTR^® SGM Plus™ multiplex system for both standard and low copy number (LCN) STR DNA analysis
Forensic Sci. Int.
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Whole genome amplification strategy for forensic genetic analysis using single or few cell equivalents of genomic DNA
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There are more references available in the full text version of this article.

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Forensic Science International

Abstract

Introduction

Section snippets

Sample preparation

DNA profiling of mixtures

Discussion

Acknowledgements

References (18)

DNA commission of the International Society of Forensic Genetics: recommendations on the interpretation of mixtures

Forensic Sci. Int.

Use of laser microdissection greatly improves the recovery of DNA from sperm on microscope slides

Forensic Sci. Int.

Single sperm cell isolation by laser microdissection

Forensic Sci. Int.

Laser microdissection and DNA typing of cells from single hair follicles

Forensic Sci. Int.

Use of fluorescence in situ hybridisation and laser microdissection to isolate male non-sperm cells in cases of sexual assault

Int. Congr. Ser.

Laser microdissection and pressure catapulting with PALM^® to assist typing of target DNA in dirt samples

Int. Congr. Ser.

A comparison of the characteristics of profiles produced with the AMPFlSTR^® SGM Plus™ multiplex system for both standard and low copy number (LCN) STR DNA analysis

Forensic Sci. Int.

Whole genome amplification strategy for forensic genetic analysis using single or few cell equivalents of genomic DNA

Anal. Biochem.

Application of low copy number DNA profiling

Croat. Med. J.

Cited by (30)

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Sperm Hy-Liter™: An effective tool for the detection of spermatozoa in sexual assault exhibits

Presence and potential of cell free DNA in different types of forensic samples

Evaluation of three DNA extraction protocols for forensic STR typing after laser capture microdissection