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Development of a one-tube extraction and amplification method for DNA analysis of sperm and epithelial cells recovered from forensic samples by laser microdissection
Forensic Science International: Genetics 6 (2012) 91–96
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Development of a one-tube extraction and amplification method for DNA analysis of sperm and epithelial cells recovered from forensic samples by laser microdissection Melanie Meredith, Jo-Anne Bright *, Sarah Cockerton, Sue Vintiner ESR, Private Bag 92021, Auckland New Zealand
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 November 2010 Received in revised form 18 January 2011 Accepted 21 February 2011
Laser microdissection can be used in forensic casework to isolate specific cell types from mixtures of biological samples. Extraction of DNA from selected cells is still required prior to STR amplification. Because of the relatively pristine nature of the recovered cells, laser microdissection is more sensitive than more traditional methods of DNA analysis, theoretically resulting in DNA profiles from less cellular material. A one-tube extraction and amplification method minimises loss of DNA through liquid transfers and reduces the potential for contamination events occurring. In this paper, the development of a one-tube method for the effective extraction of DNA from laser microdissected sperm and epithelial cells is described. The performance of the in-house method was compared to that of a commercial DNA extraction kit for extraction of DNA from sperm and the downstream compatibility with STR amplification was determined for both sperm and epithelial samples. Full IdentifilerTM profiles after 28 amplification cycles were obtained from as few as 15 epithelial cells and 30 sperm. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Laser microdissection LMD Differential extraction Sperm Epithelial cells
1. Introduction Traditionally, DNA extraction techniques such as the organic (phenol:chloroform) method are designed to facilitate both the release of DNA from the cell as well as the separation of the DNA from other components, cellular and non-cellular, present in the sample. Sexual assault evidence often consists of epithelial cells from a complainant mixed with spermatozoa (sperm cells). Therefore, the extraction method must first attempt to separate the different cell types prior to isolation of DNA from the target cells, known as differential extraction. Differential extraction methods involve a series of washes and centrifugations steps [1]. More recently, commercial kits have become available including Promega’s DifferexTM kit (Promega Corp.). Laser microdissection (LMD) is a relatively new tool used in forensic laboratories for the analysis of DNA from specifically targeted cells. It is a technique that has the potential to overcome the difficulties inherent to isolating specific cell types from mixtures of biological samples. Since its introduction to forensic laboratories, the isolation of populations of specific cell types from mixtures has been described by a variety of laboratories [2–9]. LMD employs a compound microscope to allow visualisation of the
* Corresponding author. E-mail address: [email protected] (J.-A. Bright). 1872-4973/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2011.02.007
cells and a laser for the selective capture of cells of interest, from a slide preparation containing a mixture of cells or heterogeneous tissue. A collection tube collects the dissected cells, which then undergo DNA profiling. The advent of LMD for precise cell selection provides an alternative method for the DNA profiling of forensic samples that appear to contain, or are expected to contain, low numbers of target cells. The application of LMD can reduce the potential for mixed DNA profiles occurring. Current differential extraction methodologies are not always successful at separating epithelial cells from sperm, resulting in mixed DNA profiles which can be difficult or impossible to interpret. LMD can target specific cell types when mixed with other cell types, for example, a sample that contains a few sperm in an excess of epithelial cells. A further benefit of LMD analysis includes the reduction of non-cellular PCR inhibition, providing further sensitivity to the system. A viable DNA extraction technique for cells collected by LMD is one that is able to release the DNA from the cells and also denature the cellular proteins and endogenous nucleases, which would otherwise interfere with DNA recovery and PCR. An extraction method that is also compatible with down-stream PCR analysis would be beneficial as this would allow extraction and PCR to be performed in the same tube, providing time saving benefits and improving the sensitivity of the system by minimising the loss of DNA during transfer steps, and minimising the potential for contamination.
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In this paper, the development of a one-tube method for the effective extraction and amplification of DNA from sperm and epithelial cells collected by LMD is described. The performance of the in-house method compared to a commercial DNA extraction kit Lyse-N-GoTM PCR Reagent (Pierce Chemical Co., Rockford, IL) was also assessed.
2. Method Initial experiments comparing the effectiveness of the Lyse-NGoTM and in-house extraction methods were undertaken using aliquots of diluted semen only, added directly into tubes (n = 2). Further experiments were then conducted to test the efficacy of the in-house method for the DNA analysis of sperm (n = 22), buccal and vaginal cells (n = 13) collected by LMD from cell suspensions prepared on slides. All DNA extractions were undertaken in a 0.2 mL thin-walled tube. 2.1. LMD method Sperm and epithelial cell suspensions, both mixed and neat, were fixed onto Polyethylene Napthalate (PEN) slides and stained prior to cell capture. Undiluted saliva or semen samples were transferred by direct application of 20 mL onto the slide surface using a pipette. Where necessary, cells were recovered from sterile cotton swabs by centrifugation in a spin basket (Promega Corp) after a 30 min incubation at room temperature in 400 mL extraction buffer (1.2 g L 1 tris, 3.72 g L 1 EDTA, 5.84 g L 1 NaCl, pH 8.0,). Pelleted cells were transferred to the PEN slide as for semen and saliva. Stained slides were stored at room temperature for up to a week prior to dissection. A reduced Christmas tree stain involving a 5 min stain with nuclear fast red followed by a distilled water wash and then a 5 s stain with picroindigocarmine followed by an absolute ethanol rinse was employed. Reduced times in staining were used to minimise the deleterious effect of the chemicals whilst still providing effective staining [3]. Laser microdissection was carried out using a Leica (model DM6000 B) laser microdissector. Care was taken when dissecting cells from slides so that only the cells of interest were cut from the membrane. The cutting lines were made as close to the cells as possible but without causing laser damage to the cells themselves. 2.2. Lyse-N-GoTM method 2 mL aliquots of a 1/20 dilution of semen were extracted in duplicate in a solution containing 25 mL of Lyse-N-GoTM reagent and DTT at 6 mg/mL, to a final volume of 30 mL, and incubated in the thermal cycler according to the manufacturer’s recommendations as follows: 65 8C for 30 s, 8 8C for 30 s, 65 8C for 90 s, 97 8C for 180 s, 8 8C for 60 s, 65 8C for 180 s, 97 8C for 60 s, 65 8C for 60 s, and 80 8C for 5 min (approximately 30 min total time). 2.3. In-house one-tube method 2 mL aliquots of a 1/20 dilution of semen were extracted in duplicate in a solution containing TE buffer, Tween 20 at 0.2% v/v, DTT at 0.6 mg/mL and Proteinase K (PK) at 0.1 mg/mL, to a total volume of 30 mL. Laser microdissected sperm were also extracted in TE buffer, 0.2% Tween 20, 0.6 mg/mL DTT and 0.1 mg/mL PK, to a total volume of 10 mL. Both sample types were incubated in the thermal cycler at 56 8C for 4 h. Inactivation of PK was achieved by heating at 95 8C for a further 10 min. Dissected epithelial cells were extracted using the same method except for the exclusion of DTT and a reduced incubation of 1 h at 56 8C followed by 10 min at 95 8C.
2.4. Controls Negative extraction controls were processed concurrently with each set of samples for both experiments. Positive extraction controls were extracted concurrently with samples analysed on the laser microdissector. Positive extraction controls contained a 2 mL aliquot of a 1/20 dilution of semen. In addition, amplification positive and negative controls were added to each amplification batch. 2.5. DNA analysis Prior to amplification, some samples were quantitated using the QuantifilerTM Human DNA Quantitation kit (Applied Biosystems Incorporated, Foster City, CA) on an ABI PRISM1 7500 Real-time PCR system (Applied Biosystems) according to the manufacturer’s instructions [10]. For samples from the initial comparison experiment, 0.3 ng of DNA was amplified in a separate tube at 28 cycles using the AmpFlSTR IdentifilerTM multiplex (Applied Biosystems), according to the manufacturer’s instructions, in a silver block 9700 thermal cycler (Applied Biosystems). For laser microdissected cells, the entire extract (10 mL) was amplified in the same tube that the DNA extraction was performed in using the same conditions described above (IdentifilerTM, 28 cycles, 9700 silver block). Amplified products were separated on a 3130xl Genetic Analyser (Applied Biosystems) and analysis of DNA profiles was undertaken using GeneMapperTM ID version 3.2 (Applied Biosystems) using the panels and bins provided by Applied Biosystems. A peak detection threshold of 50RFU was applied. Average peak heights were calculated, with homozygous alleles counting as one peak. 3. Results All positive and negative controls for both sets of experiments gave expected results. 3.1. Performance of the in-house one-tube method Despite sufficient amounts of DNA being detected (average 0.80 ng/mL DNA), no DNA profiling results were obtained after IdentifilerTM amplification of initial extracts of diluted semen following the in-house one-tube extraction method. This indicated that inhibitors were present in the extract preventing the successful amplification of the DNA. On further investigation, DTT was shown to be the likely cause of this inhibition. The subsequent addition of MgCl2 resulted in full IdentifilerTM profiles being obtained. A concentration of 0.5 mM MgCl2 was determined to be optimal for PCR (results not shown). The suggested mechanism for the inhibition is the reduction of Mg2+ by DTT resulting in a form of magnesium that is not usable by AmpliTaqTM Gold Polymerase. The addition of extra MgCl2 prevents the inhibition of the amplification reaction. No mixed DNA profiles were obtained from sperm and epithelial cells dissected from mixed slides indicating that the practise of dissecting only non-overlapping cells close to their boundary was successful. 3.2. Comparison of the Lyse-N-GoTM method to the in-house one-tube method The QuantifilerTM results and subsequent IdentifilerTM DNA profiles were compared for two DNA extracts of semen prepared after extraction using the Lyse-N-GoTM method and extraction using the in-house method. The quantitation results and a
M. Meredith et al. / Forensic Science International: Genetics 6 (2012) 91–96 Table 1 Comparison of QuantifilerTM and IdentifilerTM results of 2 mL of a 1/20 dilution of semen extracted using the Lyse-N-GoTM (LNG) method and the in-house method, 0.3 ng target DNA amplified. Extraction method
LNG LNG with 1 mg/mL PK In-house method
Average DNA yield (ng)
Percent alleles detected
Profile average peak height (RFU)
4.9 (n = 2) 24.9 (n = 2) 23.5 (n = 2)
56 100 100
88 261 280
93
Table 2 Comparison of number of dissected cells, average QuantifilerTM and IdentifilerTM results for sperm and epithelial samples recovered by LMD. Number of cells dissected
Theoretical yield (ng)
Average DNA yield (ng)
Average peak height (RFU)
Sperm cells
30 (n = 6) 50 (n = 16)
0.099 0.165
0.063 (s = 0.01) 0.095 (s = 0.02)
77 (s = 12) 86 (s = 14)
Epithelial cells
25 (n = 9) 50 (n = 4)
0.165 0.330
0.202 (s = 0.05) 0.384 (s = 0.04)
153 (s = 27) 281 (s = 43)
summary of profiling results obtained after these analyses are provided in Table 1. The results after the Lyse-N-GoTM method following the manufacturer’s recommended protocol with the addition of DTT were poor. The addition of PK at extraction significantly improved the performance of the method as measured by the quantitation results and average peak height after IdentifilerTM amplification. The results for the optimised Lyse-N-GoTM method were comparable to the one-tube method developed in-house. However, the in-house method has the advantage of being a simpler and cheaper technique at approximately USD$0.02 per sample versus USD$0.60 per sample with Lyse-N-GoTM. In addition, Lyse-N-GoTM is disadvantaged by a one-year expiry date from purchase. An obvious advantage of the Lyse-N-GoTM method, however, is the quick extraction time of approximately 30 min versus over 4 h for our in-house method.
The numbers of cells dissected correlated relatively well with both the quantitation result and the average peak height after IdentifilerTM amplification. The theoretical yields are calculated based on 3.3 pg of DNA per sperm and 6.6 pg per epithelial cell. The extraction of DNA from epithelial cells was the most efficient with over 100% recovery. These higher DNA yields may be partly due to inaccurate QuantifilerTM values and also possibly from the collection of nascent DNA, from ruptured epithelial cells, that were inadvertently recovered along with intact epithelial cells. Recovery of DNA from sperm was approximately 60% efficient. The lower DNA yields from sperm may also be partly explained by inaccurate QuantifilerTM values, less efficient extraction or from inhibitory effects. It is also possible that some of the collected sperm appeared morphologically intact under microscopic examination, but contained degraded DNA.
3.3. Comparison of quantitation results and STR profile quality for LMD cells extracted via the in-house one-tube method
3.4. Limits of detection for in-house one-tube method
The DNA concentration and average peak height after IdentifilerTM amplification was determined for 13 epithelial samples (buccal and vaginal cells) and 22 sperm samples recovered by LMD and extracted using the in-house method. The summary of results is in Table 2.
The limits of detection for laser microdissected sperm and epithelial cells using the in-house method were determined for IdentifilerTM. Full DNA profiles corresponding to the reference profile from the semen donor were obtained with 100 sperm (n = 4). An example electropherogram (EPG) is shown in Fig. 1. In 1 of 4 samples a full profile was obtained from 50 sperm and partial
Fig. 1. EPG of a full IdentifilerTM profile from 100 sperm.
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Fig. 2. EPG of a partial IdentifilerTM profile from 30 sperm.
profiles comprising more than 50% of alleles were obtained from as little as 30 sperm (refer Fig. 2). We therefore determined that 30 sperm was the lower limit of detection for IdentifilerTM analysis at 28 cycles. However, 150 sperm is recommended for the optimum amplification performance at 28 cycles. This number of sperm equates to approximately 0.495 ng of DNA. Although full profiles were
routinely obtained from 100 sperm, increasing the target number to 150 allows for some degradation of DNA. Lower sperm numbers can be analysed using more sensitive DNA testing, such as increasing PCR cycle numbers. This approach is not addressed in this paper. Full DNA profiles corresponding to the reference profile from the cell donors were obtained from 50 dissected vaginal cells
Fig. 3. EPG of a full IdentifilerTM profile from 50 buccal epithelial cells.
M. Meredith et al. / Forensic Science International: Genetics 6 (2012) 91–96
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Fig. 4. EPG of an IdentifilerTM profile from vaginal 15 epithelial cells.
(n = 2) and 50 dissected buccal cells (n = 2), after 28 cycles. A target of 50 epithelial cells is recommended for optimum amplification performance using 28 cycles. This equates to approximately 0.33 ng of DNA. An example EPG is in Fig. 3. The recommended limit of detection for IdentifilerTM amplification at 28 cycles is 15 epithelial cells (refer Fig. 4). Lower epithelial cell numbers can be analysed using more sensitive DNA testing. As expected, there appeared to be no difference in profiling success from the two cell types.
4. Conclusions A Lyse-N-GoTM method had previously been shown to be successful for analysis of sperm collected using LMD. Sanders et al. obtained full Profiler PlusTM (Applied Biosystems) profiles with 300 excised sperm using the manufacturer’s recommended PCR conditions of 28 cycles and a full representation of alleles for 75 and 150 sperm cells after a total of 34 PCR cycles [3]. In this study, a modified Lyse-N-GoTM method (with the addition of Proteinase K) was shown to result in higher DNA yields and higher average peak heights after amplification than the recommended protocol. The modified Lyse-N-GoTM method was comparable to an in-house extraction method that comprised TE buffer, the detergent Tween 20, and PK. The in-house extraction method was shown to be compatible to downstream PCR. The addition of DTT is necessary for the lysis of sperm. In the presence of DTT, additional Mg2+ is required to prevent inhibition of the IdentifilerTM amplification reaction. The one-tube in-house extraction and amplification method described in this paper resulted in full DNA profiles from as few as 30 sperm and 15 epithelial cells and full profiles were consistently obtained from 150 sperm and 50 epithelial cells using the manufacturer’s recommended PCR conditions of 28 cycles. Although the performance of the commercial kit Lyse-N-GoTM was comparable to that of the in-house method, the in-house
method provides added benefit as it is more cost effective (30 times cheaper than Lyse-N-GoTM) but is more time consuming. The measured quantitation result and average peak height after IdentifilerTM amplification correlated relatively well to the number of cells dissected. Quantitation of DNA is a laboratory requirement under the Quality Assurance Standards for Forensic DNA Testing Laboratories [11]. The comparison undertaken in this study has shown cell counting to be an appropriate alternative quantitation method, saving extract for analysis, minimising potential for contamination and streamlining analysis. The reduced quantitation values observed with the sperm samples in this study may have been due to some of the collected sperm containing degraded DNA. Therefore, for casework analysis, it is recommended that the optimum number of sperm collected is increased to 150 sperm to allow for this possibility. The ability to selectively target sperm using LMD enables the Forensic Scientist to report the DNA profiling results at the source level. Conventionally, if the DNA cannot be attributed to a body fluid or cell type the sub source level is reported [12]. Our laboratory undertakes LMD analysis of appropriate casework samples, such as semen stains that contain small numbers of sperm relative to the number of female epithelial cells. The criteria we use for sample selection, the success rates of analysis and general findings from our LMD casework experience will be discussed separately in a future paper. Acknowledgments The authors would like to thank Johanna Veth, Jayshree Patel and two anonymous reviewers for their helpful comments on the manuscript. References [1] P. Gill, A.J. Jeffreys, D.J. Werrett, Forensic application of DNA ‘fingerprints’, Nature 318 (6046) (1985) 577–579.
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[2] P. Pinzani, C. Orlando, M. Pazzagli, Laser-assisted microdissection for real-time PCR sample preparation, Mol. Aspects Med. 27 (2–3) (2006) 140–159. [3] C.T. Sanders, N. Sanchez, J. Ballantyne, D.A. Peterson, Laser microdissection separation of pure spermatozoa from epithelial cells for short tandem repeat analysis, J. Forensic Sci. 51 (4) (2006) 748–757. [4] M. Vandewoestyne, D. Van Hoofstat, F. Van Nieuwerburgh, D. Deforce, Automatic detection of spermatozoa for laser capture microdissection, Int. J. Leg. Med. 123 (2) (2009) 169–175. [5] B. Anoruo, R. van Oorschot, J. Mitchell, D. Howells, Isolating cells from non-sperm cellular mixtures using the PALM1 microlaser micro dissection system, Forensic Sci. Int. 173 (2–3) (2007) 93–96. [6] D. Di Martino, G. Giuffre`, N. Staiti, A. Simone, M. Le Donne, L. Saravo, Single sperm cell isolation by laser microdissection, Forensic Sci. Int. 146 (Suppl. 1) (2004) S151–S153. [7] K. Elliott, D.S. Hill, C. Lambert, T.R. Burroughes, P. Gill, Use of laser microdissection greatly improves the recovery of DNA from sperm on microscope slides, Forensic Sci. Int. 137 (1) (2003) 28–36.
[8] B.L. Lambie-Anoruo, D.V. Prince, I. Koukoulas, D.W. Howells, R.J. Mitchell, R.A.H. van Oorschot, Laser microdissection and pressure catapulting with PALM1 to assist typing of target DNA in dirt samples, Int. Congress Ser. 1288 (2006) 559– 561. [9] K. Langley, P.W. Wojtkiewicz, Application of LeicaTM laser microdissection microsystem to expedite forensic sexual assault casework, in: 16th International Symposium on Human Identification, Grapevine, Texas, 2005. [10] R.L. Green, I.C. Roinestad, C. Boland, L.K. Hennessy, Developmental validation of the QuantifilerTM real time PCR kits for the quantification of human nuclear DNA samples, J. Forensic Sci. 50 (2005) 1–17. [11] Quality assurance standards for forensic DNA testing laboratories, Forensic Sci. Commun. 2 (3) (2000) (accessed on 1 November 2010) http://www.fbi.gov/hq/ lab/fsc/backissu/july2000/codis2a.htm. [12] I.W. Evett, P.D. Gill, G. Jackson, J. Whitaker, C. Champod, Interpreting small quantities of DNA: the hierarchy of propositions and the use of Bayesian networks, J. Forensic Sci. 47 (3) (2002) 520–530.
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Development of a one-tube extraction and amplification method for DNA analysis of sperm and epithelial cells recovered from forensic samples by laser microdissection Melanie Meredith, Jo-Anne Bright *, Sarah Cockerton, Sue Vintiner ESR, Private Bag 92021, Auckland New Zealand
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 November 2010 Received in revised form 18 January 2011 Accepted 21 February 2011
Laser microdissection can be used in forensic casework to isolate specific cell types from mixtures of biological samples. Extraction of DNA from selected cells is still required prior to STR amplification. Because of the relatively pristine nature of the recovered cells, laser microdissection is more sensitive than more traditional methods of DNA analysis, theoretically resulting in DNA profiles from less cellular material. A one-tube extraction and amplification method minimises loss of DNA through liquid transfers and reduces the potential for contamination events occurring. In this paper, the development of a one-tube method for the effective extraction of DNA from laser microdissected sperm and epithelial cells is described. The performance of the in-house method was compared to that of a commercial DNA extraction kit for extraction of DNA from sperm and the downstream compatibility with STR amplification was determined for both sperm and epithelial samples. Full IdentifilerTM profiles after 28 amplification cycles were obtained from as few as 15 epithelial cells and 30 sperm. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Laser microdissection LMD Differential extraction Sperm Epithelial cells
1. Introduction Traditionally, DNA extraction techniques such as the organic (phenol:chloroform) method are designed to facilitate both the release of DNA from the cell as well as the separation of the DNA from other components, cellular and non-cellular, present in the sample. Sexual assault evidence often consists of epithelial cells from a complainant mixed with spermatozoa (sperm cells). Therefore, the extraction method must first attempt to separate the different cell types prior to isolation of DNA from the target cells, known as differential extraction. Differential extraction methods involve a series of washes and centrifugations steps [1]. More recently, commercial kits have become available including Promega’s DifferexTM kit (Promega Corp.). Laser microdissection (LMD) is a relatively new tool used in forensic laboratories for the analysis of DNA from specifically targeted cells. It is a technique that has the potential to overcome the difficulties inherent to isolating specific cell types from mixtures of biological samples. Since its introduction to forensic laboratories, the isolation of populations of specific cell types from mixtures has been described by a variety of laboratories [2–9]. LMD employs a compound microscope to allow visualisation of the
* Corresponding author. E-mail address: [email protected] (J.-A. Bright). 1872-4973/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2011.02.007
cells and a laser for the selective capture of cells of interest, from a slide preparation containing a mixture of cells or heterogeneous tissue. A collection tube collects the dissected cells, which then undergo DNA profiling. The advent of LMD for precise cell selection provides an alternative method for the DNA profiling of forensic samples that appear to contain, or are expected to contain, low numbers of target cells. The application of LMD can reduce the potential for mixed DNA profiles occurring. Current differential extraction methodologies are not always successful at separating epithelial cells from sperm, resulting in mixed DNA profiles which can be difficult or impossible to interpret. LMD can target specific cell types when mixed with other cell types, for example, a sample that contains a few sperm in an excess of epithelial cells. A further benefit of LMD analysis includes the reduction of non-cellular PCR inhibition, providing further sensitivity to the system. A viable DNA extraction technique for cells collected by LMD is one that is able to release the DNA from the cells and also denature the cellular proteins and endogenous nucleases, which would otherwise interfere with DNA recovery and PCR. An extraction method that is also compatible with down-stream PCR analysis would be beneficial as this would allow extraction and PCR to be performed in the same tube, providing time saving benefits and improving the sensitivity of the system by minimising the loss of DNA during transfer steps, and minimising the potential for contamination.
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In this paper, the development of a one-tube method for the effective extraction and amplification of DNA from sperm and epithelial cells collected by LMD is described. The performance of the in-house method compared to a commercial DNA extraction kit Lyse-N-GoTM PCR Reagent (Pierce Chemical Co., Rockford, IL) was also assessed.
2. Method Initial experiments comparing the effectiveness of the Lyse-NGoTM and in-house extraction methods were undertaken using aliquots of diluted semen only, added directly into tubes (n = 2). Further experiments were then conducted to test the efficacy of the in-house method for the DNA analysis of sperm (n = 22), buccal and vaginal cells (n = 13) collected by LMD from cell suspensions prepared on slides. All DNA extractions were undertaken in a 0.2 mL thin-walled tube. 2.1. LMD method Sperm and epithelial cell suspensions, both mixed and neat, were fixed onto Polyethylene Napthalate (PEN) slides and stained prior to cell capture. Undiluted saliva or semen samples were transferred by direct application of 20 mL onto the slide surface using a pipette. Where necessary, cells were recovered from sterile cotton swabs by centrifugation in a spin basket (Promega Corp) after a 30 min incubation at room temperature in 400 mL extraction buffer (1.2 g L 1 tris, 3.72 g L 1 EDTA, 5.84 g L 1 NaCl, pH 8.0,). Pelleted cells were transferred to the PEN slide as for semen and saliva. Stained slides were stored at room temperature for up to a week prior to dissection. A reduced Christmas tree stain involving a 5 min stain with nuclear fast red followed by a distilled water wash and then a 5 s stain with picroindigocarmine followed by an absolute ethanol rinse was employed. Reduced times in staining were used to minimise the deleterious effect of the chemicals whilst still providing effective staining [3]. Laser microdissection was carried out using a Leica (model DM6000 B) laser microdissector. Care was taken when dissecting cells from slides so that only the cells of interest were cut from the membrane. The cutting lines were made as close to the cells as possible but without causing laser damage to the cells themselves. 2.2. Lyse-N-GoTM method 2 mL aliquots of a 1/20 dilution of semen were extracted in duplicate in a solution containing 25 mL of Lyse-N-GoTM reagent and DTT at 6 mg/mL, to a final volume of 30 mL, and incubated in the thermal cycler according to the manufacturer’s recommendations as follows: 65 8C for 30 s, 8 8C for 30 s, 65 8C for 90 s, 97 8C for 180 s, 8 8C for 60 s, 65 8C for 180 s, 97 8C for 60 s, 65 8C for 60 s, and 80 8C for 5 min (approximately 30 min total time). 2.3. In-house one-tube method 2 mL aliquots of a 1/20 dilution of semen were extracted in duplicate in a solution containing TE buffer, Tween 20 at 0.2% v/v, DTT at 0.6 mg/mL and Proteinase K (PK) at 0.1 mg/mL, to a total volume of 30 mL. Laser microdissected sperm were also extracted in TE buffer, 0.2% Tween 20, 0.6 mg/mL DTT and 0.1 mg/mL PK, to a total volume of 10 mL. Both sample types were incubated in the thermal cycler at 56 8C for 4 h. Inactivation of PK was achieved by heating at 95 8C for a further 10 min. Dissected epithelial cells were extracted using the same method except for the exclusion of DTT and a reduced incubation of 1 h at 56 8C followed by 10 min at 95 8C.
2.4. Controls Negative extraction controls were processed concurrently with each set of samples for both experiments. Positive extraction controls were extracted concurrently with samples analysed on the laser microdissector. Positive extraction controls contained a 2 mL aliquot of a 1/20 dilution of semen. In addition, amplification positive and negative controls were added to each amplification batch. 2.5. DNA analysis Prior to amplification, some samples were quantitated using the QuantifilerTM Human DNA Quantitation kit (Applied Biosystems Incorporated, Foster City, CA) on an ABI PRISM1 7500 Real-time PCR system (Applied Biosystems) according to the manufacturer’s instructions [10]. For samples from the initial comparison experiment, 0.3 ng of DNA was amplified in a separate tube at 28 cycles using the AmpFlSTR IdentifilerTM multiplex (Applied Biosystems), according to the manufacturer’s instructions, in a silver block 9700 thermal cycler (Applied Biosystems). For laser microdissected cells, the entire extract (10 mL) was amplified in the same tube that the DNA extraction was performed in using the same conditions described above (IdentifilerTM, 28 cycles, 9700 silver block). Amplified products were separated on a 3130xl Genetic Analyser (Applied Biosystems) and analysis of DNA profiles was undertaken using GeneMapperTM ID version 3.2 (Applied Biosystems) using the panels and bins provided by Applied Biosystems. A peak detection threshold of 50RFU was applied. Average peak heights were calculated, with homozygous alleles counting as one peak. 3. Results All positive and negative controls for both sets of experiments gave expected results. 3.1. Performance of the in-house one-tube method Despite sufficient amounts of DNA being detected (average 0.80 ng/mL DNA), no DNA profiling results were obtained after IdentifilerTM amplification of initial extracts of diluted semen following the in-house one-tube extraction method. This indicated that inhibitors were present in the extract preventing the successful amplification of the DNA. On further investigation, DTT was shown to be the likely cause of this inhibition. The subsequent addition of MgCl2 resulted in full IdentifilerTM profiles being obtained. A concentration of 0.5 mM MgCl2 was determined to be optimal for PCR (results not shown). The suggested mechanism for the inhibition is the reduction of Mg2+ by DTT resulting in a form of magnesium that is not usable by AmpliTaqTM Gold Polymerase. The addition of extra MgCl2 prevents the inhibition of the amplification reaction. No mixed DNA profiles were obtained from sperm and epithelial cells dissected from mixed slides indicating that the practise of dissecting only non-overlapping cells close to their boundary was successful. 3.2. Comparison of the Lyse-N-GoTM method to the in-house one-tube method The QuantifilerTM results and subsequent IdentifilerTM DNA profiles were compared for two DNA extracts of semen prepared after extraction using the Lyse-N-GoTM method and extraction using the in-house method. The quantitation results and a
M. Meredith et al. / Forensic Science International: Genetics 6 (2012) 91–96 Table 1 Comparison of QuantifilerTM and IdentifilerTM results of 2 mL of a 1/20 dilution of semen extracted using the Lyse-N-GoTM (LNG) method and the in-house method, 0.3 ng target DNA amplified. Extraction method
LNG LNG with 1 mg/mL PK In-house method
Average DNA yield (ng)
Percent alleles detected
Profile average peak height (RFU)
4.9 (n = 2) 24.9 (n = 2) 23.5 (n = 2)
56 100 100
88 261 280
93
Table 2 Comparison of number of dissected cells, average QuantifilerTM and IdentifilerTM results for sperm and epithelial samples recovered by LMD. Number of cells dissected
Theoretical yield (ng)
Average DNA yield (ng)
Average peak height (RFU)
Sperm cells
30 (n = 6) 50 (n = 16)
0.099 0.165
0.063 (s = 0.01) 0.095 (s = 0.02)
77 (s = 12) 86 (s = 14)
Epithelial cells
25 (n = 9) 50 (n = 4)
0.165 0.330
0.202 (s = 0.05) 0.384 (s = 0.04)
153 (s = 27) 281 (s = 43)
summary of profiling results obtained after these analyses are provided in Table 1. The results after the Lyse-N-GoTM method following the manufacturer’s recommended protocol with the addition of DTT were poor. The addition of PK at extraction significantly improved the performance of the method as measured by the quantitation results and average peak height after IdentifilerTM amplification. The results for the optimised Lyse-N-GoTM method were comparable to the one-tube method developed in-house. However, the in-house method has the advantage of being a simpler and cheaper technique at approximately USD$0.02 per sample versus USD$0.60 per sample with Lyse-N-GoTM. In addition, Lyse-N-GoTM is disadvantaged by a one-year expiry date from purchase. An obvious advantage of the Lyse-N-GoTM method, however, is the quick extraction time of approximately 30 min versus over 4 h for our in-house method.
The numbers of cells dissected correlated relatively well with both the quantitation result and the average peak height after IdentifilerTM amplification. The theoretical yields are calculated based on 3.3 pg of DNA per sperm and 6.6 pg per epithelial cell. The extraction of DNA from epithelial cells was the most efficient with over 100% recovery. These higher DNA yields may be partly due to inaccurate QuantifilerTM values and also possibly from the collection of nascent DNA, from ruptured epithelial cells, that were inadvertently recovered along with intact epithelial cells. Recovery of DNA from sperm was approximately 60% efficient. The lower DNA yields from sperm may also be partly explained by inaccurate QuantifilerTM values, less efficient extraction or from inhibitory effects. It is also possible that some of the collected sperm appeared morphologically intact under microscopic examination, but contained degraded DNA.
3.3. Comparison of quantitation results and STR profile quality for LMD cells extracted via the in-house one-tube method
3.4. Limits of detection for in-house one-tube method
The DNA concentration and average peak height after IdentifilerTM amplification was determined for 13 epithelial samples (buccal and vaginal cells) and 22 sperm samples recovered by LMD and extracted using the in-house method. The summary of results is in Table 2.
The limits of detection for laser microdissected sperm and epithelial cells using the in-house method were determined for IdentifilerTM. Full DNA profiles corresponding to the reference profile from the semen donor were obtained with 100 sperm (n = 4). An example electropherogram (EPG) is shown in Fig. 1. In 1 of 4 samples a full profile was obtained from 50 sperm and partial
Fig. 1. EPG of a full IdentifilerTM profile from 100 sperm.
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Fig. 2. EPG of a partial IdentifilerTM profile from 30 sperm.
profiles comprising more than 50% of alleles were obtained from as little as 30 sperm (refer Fig. 2). We therefore determined that 30 sperm was the lower limit of detection for IdentifilerTM analysis at 28 cycles. However, 150 sperm is recommended for the optimum amplification performance at 28 cycles. This number of sperm equates to approximately 0.495 ng of DNA. Although full profiles were
routinely obtained from 100 sperm, increasing the target number to 150 allows for some degradation of DNA. Lower sperm numbers can be analysed using more sensitive DNA testing, such as increasing PCR cycle numbers. This approach is not addressed in this paper. Full DNA profiles corresponding to the reference profile from the cell donors were obtained from 50 dissected vaginal cells
Fig. 3. EPG of a full IdentifilerTM profile from 50 buccal epithelial cells.
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Fig. 4. EPG of an IdentifilerTM profile from vaginal 15 epithelial cells.
(n = 2) and 50 dissected buccal cells (n = 2), after 28 cycles. A target of 50 epithelial cells is recommended for optimum amplification performance using 28 cycles. This equates to approximately 0.33 ng of DNA. An example EPG is in Fig. 3. The recommended limit of detection for IdentifilerTM amplification at 28 cycles is 15 epithelial cells (refer Fig. 4). Lower epithelial cell numbers can be analysed using more sensitive DNA testing. As expected, there appeared to be no difference in profiling success from the two cell types.
4. Conclusions A Lyse-N-GoTM method had previously been shown to be successful for analysis of sperm collected using LMD. Sanders et al. obtained full Profiler PlusTM (Applied Biosystems) profiles with 300 excised sperm using the manufacturer’s recommended PCR conditions of 28 cycles and a full representation of alleles for 75 and 150 sperm cells after a total of 34 PCR cycles [3]. In this study, a modified Lyse-N-GoTM method (with the addition of Proteinase K) was shown to result in higher DNA yields and higher average peak heights after amplification than the recommended protocol. The modified Lyse-N-GoTM method was comparable to an in-house extraction method that comprised TE buffer, the detergent Tween 20, and PK. The in-house extraction method was shown to be compatible to downstream PCR. The addition of DTT is necessary for the lysis of sperm. In the presence of DTT, additional Mg2+ is required to prevent inhibition of the IdentifilerTM amplification reaction. The one-tube in-house extraction and amplification method described in this paper resulted in full DNA profiles from as few as 30 sperm and 15 epithelial cells and full profiles were consistently obtained from 150 sperm and 50 epithelial cells using the manufacturer’s recommended PCR conditions of 28 cycles. Although the performance of the commercial kit Lyse-N-GoTM was comparable to that of the in-house method, the in-house
method provides added benefit as it is more cost effective (30 times cheaper than Lyse-N-GoTM) but is more time consuming. The measured quantitation result and average peak height after IdentifilerTM amplification correlated relatively well to the number of cells dissected. Quantitation of DNA is a laboratory requirement under the Quality Assurance Standards for Forensic DNA Testing Laboratories [11]. The comparison undertaken in this study has shown cell counting to be an appropriate alternative quantitation method, saving extract for analysis, minimising potential for contamination and streamlining analysis. The reduced quantitation values observed with the sperm samples in this study may have been due to some of the collected sperm containing degraded DNA. Therefore, for casework analysis, it is recommended that the optimum number of sperm collected is increased to 150 sperm to allow for this possibility. The ability to selectively target sperm using LMD enables the Forensic Scientist to report the DNA profiling results at the source level. Conventionally, if the DNA cannot be attributed to a body fluid or cell type the sub source level is reported [12]. Our laboratory undertakes LMD analysis of appropriate casework samples, such as semen stains that contain small numbers of sperm relative to the number of female epithelial cells. The criteria we use for sample selection, the success rates of analysis and general findings from our LMD casework experience will be discussed separately in a future paper. Acknowledgments The authors would like to thank Johanna Veth, Jayshree Patel and two anonymous reviewers for their helpful comments on the manuscript. References [1] P. Gill, A.J. Jeffreys, D.J. Werrett, Forensic application of DNA ‘fingerprints’, Nature 318 (6046) (1985) 577–579.
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[2] P. Pinzani, C. Orlando, M. Pazzagli, Laser-assisted microdissection for real-time PCR sample preparation, Mol. Aspects Med. 27 (2–3) (2006) 140–159. [3] C.T. Sanders, N. Sanchez, J. Ballantyne, D.A. Peterson, Laser microdissection separation of pure spermatozoa from epithelial cells for short tandem repeat analysis, J. Forensic Sci. 51 (4) (2006) 748–757. [4] M. Vandewoestyne, D. Van Hoofstat, F. Van Nieuwerburgh, D. Deforce, Automatic detection of spermatozoa for laser capture microdissection, Int. J. Leg. Med. 123 (2) (2009) 169–175. [5] B. Anoruo, R. van Oorschot, J. Mitchell, D. Howells, Isolating cells from non-sperm cellular mixtures using the PALM1 microlaser micro dissection system, Forensic Sci. Int. 173 (2–3) (2007) 93–96. [6] D. Di Martino, G. Giuffre`, N. Staiti, A. Simone, M. Le Donne, L. Saravo, Single sperm cell isolation by laser microdissection, Forensic Sci. Int. 146 (Suppl. 1) (2004) S151–S153. [7] K. Elliott, D.S. Hill, C. Lambert, T.R. Burroughes, P. Gill, Use of laser microdissection greatly improves the recovery of DNA from sperm on microscope slides, Forensic Sci. Int. 137 (1) (2003) 28–36.
[8] B.L. Lambie-Anoruo, D.V. Prince, I. Koukoulas, D.W. Howells, R.J. Mitchell, R.A.H. van Oorschot, Laser microdissection and pressure catapulting with PALM1 to assist typing of target DNA in dirt samples, Int. Congress Ser. 1288 (2006) 559– 561. [9] K. Langley, P.W. Wojtkiewicz, Application of LeicaTM laser microdissection microsystem to expedite forensic sexual assault casework, in: 16th International Symposium on Human Identification, Grapevine, Texas, 2005. [10] R.L. Green, I.C. Roinestad, C. Boland, L.K. Hennessy, Developmental validation of the QuantifilerTM real time PCR kits for the quantification of human nuclear DNA samples, J. Forensic Sci. 50 (2005) 1–17. [11] Quality assurance standards for forensic DNA testing laboratories, Forensic Sci. Commun. 2 (3) (2000) (accessed on 1 November 2010) http://www.fbi.gov/hq/ lab/fsc/backissu/july2000/codis2a.htm. [12] I.W. Evett, P.D. Gill, G. Jackson, J. Whitaker, C. Champod, Interpreting small quantities of DNA: the hierarchy of propositions and the use of Bayesian networks, J. Forensic Sci. 47 (3) (2002) 520–530.