Forensic Science International: Genetics
Volume 6, Issue 1 , Pages 1-16, January 2012

Development of a rapid, 96-well alkaline based differential DNA extraction method for sexual assault evidence

California Department of Justice Jan Bashinski DNA Laboratory, 1001 W. Cutting Blvd., Suite 110, Richmond, CA 94804, USA

Received 23 July 2010; received in revised form 15 December 2010; accepted 20 December 2010. published online 03 February 2011.

Article Outline

Abstract 

We present a rapid alkaline lysis procedure for the extraction of DNA from sexual assault evidence that generates purified sperm fraction extracts that yield STR typing results similar to those obtained from the traditional organic/dithiothreitol differential extraction. Specifically, a sodium hydroxide based differential extraction method has been developed in a single-tube format and further optimized in a 96-well format. The method yields purified extracts from a small sample set (∼2–6 swabs) in approximately 2h and from a larger sample set (up to 96 swabs) in approximately 4h. While conventional differential extraction methods require vigorous sample manipulation to remove the spermatozoa from the substrate, the method described here exploits the propensity of sperm to adhere to a substrate and does not require any manipulation of the substrate after it is sampled. For swabs, sample handling is minimized by employing a process where the tip of the swab, including the shaft, is transferred to the appropriate vessel eliminating the need for potentially hazardous scalpels to separate the swab material from the shaft. The absence of multiple handling steps allows the process to be semi-automated, however the procedure as described here does not require use of a robotic system. This method may provide forensic laboratories a cost-effective tool for the eradication of backlogs of sexual assault evidence, and more timely service to their client agencies. In addition, we have demonstrated that a modification of the procedure can be used to retrieve residual sperm-cell DNA from previously extracted swabs.

Keywords: Forensic sciences, Alkaline lysis, Sodium hydroxide, DNA typing, Differential extraction, Sexual assault evidence

 

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1. Introduction 

Escalating demands on forensic laboratories coupled with laborious methods used in the analysis of sexual assault evidence have contributed to an increasing backlog of evidence from sexual assault cases. As a result, the ability to examine sexual assault evidence immediately after the event is a capability few laboratories have. This is especially true for suspectless sexual assaults, despite the ability to search national databases like CODIS. The need to increase throughput in DNA typing laboratories has generated a great deal of interest in alterative DNA extraction procedures, including alkaline lysis procedures with blood, semen, saliva [1], [2], fingernails [3], head hair [4] and formalin fixed paraffin embedded tissue [5]. The benefits of an alkaline lysis extraction method include a simple procedure with decreased extraction time and minimal reagent costs. This would seem to make alkaline lysis a perfect tool for decreasing the backlog of evidence collected in sexual assault cases [6]. However, the more complex nature of sexual assault evidence, that typically includes mixtures of spermatozoa and female epithelial cells, prevents a simple, single-step alkaline lysis procedure from being used with these types of samples. Traditionally, the separation of sperm cell DNA from non-sperm cell DNA has been achieved by a differential extraction method [7], in which non-sperm cells are first lysed with a sodium dodecyl sulfate (SDS)/proteinase K solution. The intact sperm cells are separated from the non-sperm cell lysate using a series of centrifugation and wash steps, after which the sperm cell portion is lysed in a SDS/proteinase K/DTT solution. In the last step of the differential extraction procedure, the non-sperm-cell and sperm-cell lysates are extracted independently to achieve final separated fractions. While the efficiency of this last step in the differential extraction procedure has improved due to the use of automated DNA extraction methods, the initial steps required to separate the non-sperm and sperm cell fractions have been less amenable to automation and typically rely on manual processes that are labor and time intensive [8], [9], [10], [11], [12]. Furthermore, the traditional method is dependent on the removal of the spermatozoa from the substrate prior to their lysis, which has been estimated to yield 10–40% recoveries of the spermatozoa from the substrates [13], [14]. Alternative methods of generating profiles from sperm cell DNA contained in sexual assault evidence include modified differential extraction methods [15], [16], [17], [18], [19], [20], [21] and laser microdissection [22], but each is dependent on the removal of the sperm cells from the substrate prior to sperm cell lysis.

In this article, we report the development of an alkaline differential extraction method that rapidly yields purified sperm fraction lysates with minimal sample handling. Non-sperm lysates are generated by heating the substrate in a mild alkaline solution (0.1N NaOH), followed by neutralization and enzymatic digestion steps to remove residual non-sperm DNA from the substrate. Sperm fraction lysates are subsequently generated by heating the substrate in 1N NaOH, which are then concentrated and purified using silica columns. This method was developed using mock sexual assault evidence swabs that were prepared by transferring semen onto buccal swabs collected from a female contributor, and has been successfully used with authentic post-coital swabs that have been stored frozen as indicated by the California Medical Protocol for Examination of Sexual Assault and Child Abuse Victims [23]. Additionally, swabs that have previously been extracted using a standard differential extraction method have been successfully re-extracted with this alkaline lysis procedure.

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2. Materials and methods 

2.1. Samples 

Post-coital swabs, semen and saliva samples were donated by laboratory staff. Pre-quantified, high molecular weight, human genomic DNA was obtained from Promega, Madison, WI (Male-#G1471), Applied Biosystems, Foster City, CA (TaqMan® Control DNA) and ATCC, Manassas, VA (HL60).

2.2. Preparation and sampling of mock sexual assault evidence swabs 

Simulated sexual assault evidence (SAE) swabs were prepared by transferring semen onto buccal swabs collected from female contributors using sterile cotton swabs (Puritan Medical Products, Guilford, ME), which were allowed to air-dry at room temperature for a period of 2–4h prior to storing frozen at −20°C [23]. All SAE swabs were sampled by cutting the tip off with dissecting scissors or breaking the tip off with a clean, gloved hand and/or a Kimwipe to yield a section of the swab tip and shaft that was less than or equal to ∼1.5cm in length.

2.3. Alkaline lysis 

All alkaline lysis steps were conducted in dry bath incubators with NaOH solutions prepared by dissolving NaOH pellets (Sigma, St. Louis, MO) in DEPC treated molecular biology grade water (Rockland Immunochemicals, Gilbertsville, PA) or sterile water. Single-tube (1.5mL microcentrifuge tube (Dynalab, Rochester, NY)), lysis steps were carried out in VWR Heat Block 1 incubators (VWR, West Chester, PA) with 10mm heat block inserts as indicated in Fig. 1a. 96-Well lysis steps were conducted in Boekel Digital Dry Bath Incubators (Boekel Scientific, Festerville, PA) fitted with heat block inserts (Promega) and custom, VP 741 I, 96-well plate heating block inserts (V&P Scientific, San Diego, CA) using color coded Abgene AB-0932, 2.2mL, 96-well plates (ThermoFisher Scientific, Pittsburgh, PA) for each step as indicated in Fig. 1a.

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  • Fig. 1. 

    (a) Flow-chart depicting optimized single-tube (left) and 96-well (right) alkaline differential extraction methods as indicated by parameter sets 22 and 28 in Table 2, Table 3, respectively. Note: A maximum of 2–3 samples should be extracted at one time to prevent exceeding the intended lysis times while transferring substrates to Spin-X inserts. (b) Flow-chart depicting sperm fraction concentration and clean-up steps for single-tube (left) and 96-well (right) alkaline differential extraction methods as indicated by parameter sets 22 and 28 in Table 2, Table 3, respectively. (c) Flow-chart depicting non-sperm fraction concentration and clean-up steps Note: The volume of glacial acetic acid used to adjust the pH of the non-sperm lysates from the single-tube alkaline extraction should be reduced to 2.7μL to compensate for the lower non-sperm lysate volume in the single-tube method (∼386μL) versus ∼709μL for the 96-well method.

2.4. DNase digestion 

DNase digestion was completed with DNase I (Invitrogen, Carlsbad, CA) in a 1× DNase I Reaction Buffer (20mM Tris–HCl (pH 8), 2mM MgCl2, 50mM KCl) as indicated by the manufacturer.

Specifically, each reaction was carried out at 37°C in dry bath incubators for 1–1½h in a solution consisting of 0.1U/μL DNase I and 10× DNase I Reaction Buffer diluted in DEPC-treated molecular biology grade water to bring the solution to a 1× reaction buffer concentration.

2.5. Concentration and clean-up of lysates 

Sperm fraction lysates were concentrated and cleaned up by microdialysis filtration with Centricon® YM-100 devices (Millipore, Billerica, MA) or by silica-binding with NucleoSpin XS columns or NucleoSpin-96 tissue plates (Macherey-Nagel, Bethleham, PA), which are referred to as “C100”, “NSXS” and “NS96T”, respectively, in Table 2, Table 3. Centricon® YM-100 devices were used following neutralization with 2M Tris base (pH 7.5) and dilution in TE−4 buffer (10mM Tris, pH 7.5; 0.1mM EDTA) with repeated TE−4 buffer rinses, as indicated in Table 2. NucleoSpin XS columns/NucleoSpin-96 tissue plates were loaded after neutralization with glacial acetic acid, dilution of the lysates in TE−4 buffer in 2mL microcentrifuge tubes/4mL 96-well pyramid bottom plates (E&K Scientific, Santa Clara, CA) and the addition of NT or NTC binding buffer (provided by Macherey-Nagel). The silica columns/96-well plates were washed with ethanol based B5 buffer (provided by Macherey-Nagel) with the waste collected in 2mL microcentrifuge tubes/1.1mL, 96 deep well plates (E&K Scientific). The DNA was eluted with TE−4, Tris, BE elution buffer (provided by Macherey-Nagel), or sterile H2O into capless 1.5mL microcentrifuge tubes/1/2 skirt PCR plates (E&K Scientific) and heated in 90°C dry bath incubators to drive off residual ethanol and further concentrate the samples, as indicated in Table 2, Table 3 and indicated in Fig. 1b.

Non-sperm fraction lysates generated in these studies did not require concentration. However, these samples have been successfully concentrated and purified using microdialysis devices (as indicated above) and silica-binding devices (as described in Fig. 1c), for samples containing few non-sperm cells or where premature lysis of the spermatozoa occurred. Additionally, the NucleoFast 96-well microdialysis plates (Macherey-Nagel) have been successfully employed with neutralized non-sperm fraction lysates and repeated TE−4 buffer rinses.

2.6. Standard procedure for differential extraction of mock sexual assault evidence swabs 

Standard differential extractions of sperm and non-sperm fractions were performed on swab substrates that had been excised from the shaft with sterile scalpels using a dithiothreitol-based digestion protocol (DTT) [7] with Centricon® YM-100 concentration of the aqueous layer from the phenol/chloroform extraction.

2.7. DNA quantification 

Extracts were quantified with a previously in-house developed quadruplex qPCR assay (nuTH01–nuSRY–nuCSF–IPC) on an Applied Biosystems 7500 Real Time PCR system (7500 SDS software v 1.3) [24].

2.8. STR genotyping 

The AmpFlSTR® Identifiler™ PCR Amplification kit (Applied Biosystems) was used for STR genotyping. Unless otherwise noted and whenever possible, 1ng template quantities of autosomal DNA were targeted in the 25μL PCRs that were performed according to vendor instructions on a GeneAmp® 9700 PCR thermal cycler (Applied Biosystems). STRs were resolved and detected on an ABI Prism® 3130 Genetic Analyzer (Applied Biosystems) according to vendor instructions. Electrophoresis data were analyzed using GeneMapper™ ID 3.2 (Applied Biosystems) using 50 RFU analytical threshold.

2.9. Experimental setup 

Development of the alkaline differential extraction method was conducted in two phases. The first phase was conducted in a single-tube (1.5mL microcentrifuge tube) format and included the determination of the optimal epithelial cell and spermatozoa lysis conditions. The second phase was conducted in a 96-well format using the Slicprep™ 96 devices (Promega) with modification of the single tube lysis conditions as required. Additionally, data from the standard differential extraction was generated from mock sexual assault swabs containing, 1, 0.1 and 0.01μL semen for comparison purposes.

2.9.1. Determination of initial lysis conditions for lysis of epithelial cells in the presence of semen, spermatozoa, and the development of a single-tube (1.5mL) alkaline differential extraction 

The first step in the development of the alkaline differential extraction method was the determination of the minimum concentration of NaOH required to lyse epithelial cells in the presence of semen. Semen is known to have a buffering capacity, and previous alkaline lysis methods for the extraction of DNA from semen stains had reported NaOH concentrations that varied by a factor of two [1], [2]. In addition, the concentration of NaOH required to lyse spermatozoa absent of seminal fluid was determined, using microscopy to verify the absence of the cells targeted by the alkaline lysis. The use of microscopy to determine the initial lysis conditions for the non-sperm lysis step was preferred over a quantitative PCR (qPCR) assay, as the presence of male DNA is expected from lysed white blood cells contained in semen, which can exceed 1,000,000/mL [25]. Therefore, the use of qPCR data to infer premature lysis of spermatozoa during the non-sperm lysis step was not attempted and empirical determination of an optimized set of extraction parameters capable of yielding results similar to the standard differential extraction method was conducted as indicated below, and described in Table 2, Table 3.

An epithelial cell suspension was prepared from a buccal swab by pelleting the epithelial cells by centrifugation in sterile water and mixing in a final volume of ∼80μL. 10μL aliquots were transferred to seven tubes, each containing 20μL of neat semen. These mixtures were heated at 75°C in 80μL of 0.05, 0.1, 0.2, 0.4, 0.8, 1.0N NaOH or sterile water as a control for 5min, and neutralized with 2M Tris (pH 7.5) as indicated in Table 1. The remaining cellular material was pelleted by centrifugation and rinsed in 400μL of phosphate buffered saline (PBS) with a final cellular suspension of ∼50μL. 10μL of this suspension was transferred to a glass slide, heat fixed, “Christmas tree” stained, and examined with brightfield microscopy at 400× [26]. A second set of samples was treated in the same way with lysis at 95°C in 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7N NaOH solutions or sterile water as a control.

Table 1. Summary of results for epithelial cell and spermatozoa lysis experiments.
Sample typeCells targeted for lysisLysis temperature (°C)NaOH concentration (N)Volume of 2M Tris pH 7.5 used in neutralization (μL)Epithelial cells observed with 400× microscopySpermatozoa observed with 400× microscopy
Epithelial cell/semen mixtureEpithelial cells750.053.6NumerousNumerous
Epithelial Cell/Semen MixtureEpithelial cells750.17.2NumerousNumerous
Epithelial cell/semen mixtureEpithelial cells750.214.4NumerousNumerous
Epithelial cell/semen mixtureEpithelial cells750.428.8Few possibleNumerous
Epithelial cell/semen mixtureEpithelial cells750.857.6One possibleFew
Epithelial cell/semen mixtureEpithelial cells751.072NoneNone
Epithelial cell/semen mixtureEpithelial cells7500NumerousNumerous

Epithelial cell/semen mixtureEpithelial cells950.17.2Few possibleNumerous
Epithelial cell/semen mixtureEpithelial cells950.214.4Few possibleNumerous
Epithelial cell/semen mixtureEpithelial cells950.321.6NoneNumerous
Epithelial cell/semen mixtureEpithelial cells950.428.8NoneFew
Epithelial cell/semen mixtureEpithelial cells950.536NoneFew
Epithelial cell/semen mixtureEpithelial cells950.643.2NoneFew
Epithelial cell/semen mixtureEpithelial cells950.750.4NoneFew
Epithelial cell/semen mixtureEpithelial cells9500NumerousNumerous

Rinsed spermatozoaSpermatozoa750.0536N/ANumerous
Rinsed spermatozoaSpermatozoa750.172N/ANumerous
Rinsed spermatozoaSpermatozoa750.2144N/ANumerous
Rinsed spermatozoaSpermatozoa750.4288N/ANone
Rinsed spermatozoaSpermatozoa750.8576N/AOne
Rinsed spermatozoaSpermatozoa751.0720N/ANone

Note: Lysis conditions deemed to be appropriate for use as a starting point in the alkaline differential extraction are underlined in bold font.

The concentration of NaOH required for 75°C lysis of spermatozoa absent of the buffering seminal fluid was determined by lysing rinsed spermatozoa under conditions similar to those present after alkaline lysis of the epithelial cells and digestion of residual non-sperm DNA by the DNase. Specifically, a rinsed spermatozoa solution was prepared by mixing 100μL of neat semen with 400μL of sterile water, pelleting the spermatozoa by centrifugation, carefully removing ∼400μL of the supernatant, repeating once, and suspending the spermatozoa in a final volume of ∼100μL. 10μL aliquots of the rinsed spermatozoa were transferred to each of seven 1.5mL microcentrifuge tubes. To simulate conditions where the buffering seminal fluid has previously been removed from a swab, cotton swabs that had been wetted with 0.4N NaOH were centrifuged using Spin-X inserts (Corning, Lowell, MA), wetted with 1× DNase buffer, centrifuged using Spin-X inserts (Corning), and added to each tube. Addition of 400μL of 0.05, 0.1, 0.2, 0.4, 0.8, 1.0N NaOH preceded lysis at 75°C for 5min with subsequent centrifuging of the substrates in Spin-X inserts (Corning). Each solution was neutralized with 2M Tris (pH 7.5) as indicated in Table 1, and rinsed twice with 400μL of phosphate buffered saline using centrifugation to pellet the cellular material. The cellular pellet was resuspended in a final volume of ∼50μL and a 10μL aliquot was transferred to a glass slide, heat-fixed, Christmas tree stained and examined with 400× brightfield microscopy.

Initially, development of the single-tube alkaline differential extraction included three steps based on the results from the epithelial cell lysis and spermatozoa lysis experiments, as well as previous experience with DNase digestion. Specifically, epithelial cell lysis was conducted by transferring the entire tip of the swab, including the shaft, to a 1.5mL microcentrifuge tube containing 400μL of 0.4N NaOH and heating at 75°C for 5min, with subsequent centrifugation of the substrate in Spin-X inserts (Corning) for 30s in a microcentrifuge (Tomy, Fremont, CA) at 11,000rpm. Residual non-sperm DNA was removed by transferring the substrate to a separate tube containing 400μL of 0.1U/μL DNase I in 1× DNase I Reaction Buffer and heating for 1h at 37°C, with subsequent centrifugation of the substrate in Spin-X inserts (Corning) at 11,000rpm for 30s. Finally, simultaneous inactivation of DNase and lysis of the spermatozoa was completed by immersing the substrate in 400μL of 1N NaOH and heating at 75°C for 5min, with subsequent centrifugation of the substrate in Spin-X inserts (Corning) at 11,000rpm for 30s to maximize DNA recoveries. These parameters were modified and optimized by empirical testing with replicate (duplicates or triplicates) mock SAE swabs prepared from a single semen contributor and female buccal contributor to minimize variation from the sample sources. The optimized single-tube alkaline differential extraction parameters, parameter set 22, include a 1× buffer (DNase) rinse prior to the DNase digestion step as indicated in Table 2. When necessary, sperm fraction lysates were concentrated and purified with microdialysis devices or silica columns. Silica columns were the preferred method due to the presence of co-extracted PCR inhibitors in the sperm fraction lysates (as indicated in Column L of Table 2), and their ability to generate purified and concentrated lysates in less than 1h.

Table 2. Parameters used in the development of the single-tube alkaline differential extraction method.
ABCDEFGHIJKLM
Parameter setSemen (μL)Non-sperm lysis NaOH (N)Non-sperm lysis temp (°C/min)Room temp neutralization (min)DNase digestion 37°C (min)Sperm lysis tempa (°C/min)Sperm fraction clean-up methodLoading buffer (type/mL)Wash steps (type/mL/#)Elution steps (μLTE−4/#)Identifiler™ results
MajorbMinorb
10.10.475/5–6075/10C100TE−4/0.8TE−4/1/2N/AFemaleMale
11.00.475/5–6075/10C100TE−4/0.8TE−4/1/2N/AFemaleMale
1100.475/5–6075/10C100TE−4/0.8TE−4/1/2N/AFemaleMale
11000.475/5–6075/10C100TE−4/0.8TE−4/1/2N/AMaleFemale
21.00.475/10–6075/10C100TE−4/0.8TE−4/1/2N/AFemaleMale
31.00.875/5–6075/10C100TE−4/0.8TE−4/1/2N/AFemaleMale
41.00.875/10–6075/10C100TE−4/0.8TE−4/1/2N/AFemaleMale
51.00.175/10–6075/10C100TE−4/0.8TE−4/1/2N/AFemaleMale
61.00.275/10–6075/10C100TE−4/0.8TE−4/1/2N/AFemaleMale
71.00.175/5–6075/10C100TE−4/0.8TE−4/1/2N/AMaleFemale
81.00.195/10–6075/10C100TE−4/0.8TE−4/1/2N/AMaleFemale
91.00.295/5–6075/10C100TE−4/0.8TE−4/1/2N/AMaleFemale
101.00.295/10–6075/10C100TE−4/0.8TE−4/1/2N/AMaleFemale
111.00.195/356075/10C100TE−4/0.8TE−4/1/2N/AMaleNone
121.00.195/556075/10C100TE−4/0.8TE−4/1/2N/AMaleNone
131.00.195/156075/10C100TE−4/0.8TE−4/1/2N/AMaleNone
141.00.195/256075/10C100TE−4/0.8TE−4/1/2N/AMaleNone
140.10.195/256075/10C100TE−4/0.8TE−4/1/2N/AMaleNone
140.010.195/256075/10C100TE−4/0.8TE−4/1/2N/AMale, inhibitedFemale
151.00.195/356075/10C100TE−4/0.8TE−4/1/2N/AMaleNone
161.00.195/256075/10NSXSNT/0.1B5/0.1/110/2MaleFemale
170.010.195/2512075/5C100TE−4/1.6TE−4/2/2N/AMale, inhibitedFemale
180.10.195/256075/10NSXSTE−4/0.775 & NTC/0.3B5/0.1/110/2MaleFemale
180.010.195/256075/10NSXSTE−4/0.775 & NTC/0.3B5/0.1/110/2FemaleND
190.10.195/256075/10NSXSTE−4/0.775 & NTC/0.3B5/0.1/120c/1dMaleFemale
190.010.195/256075/10NSXSTE−4/0.775 & NTC/0.3B5/0.1/120c/1dFemaleMale
200.10.195/256075/2NSXSTE−4/0.775 & NTC/0.3B5/0.1/210/2Male, inhibitedND
210.10.195/256075/5NSXSTE−4/0.775 & NTC/0.3B5/0.1/210/2Male, inhibitedND
22e0.10.195/256075/2NSXSTE−4/0.775 & NTC/0.3B5/0.05/210/2MaleFemale

aNote: All sperm lysis steps conducted with 1N NaOH.

bNote: Only the sperm cell fractions are being assessed for the presence of male and female alleles.

cTE−4 heated to 70°C prior to use as elution buffer.

dNSXS column heated in 70°C incubator 10min prior to elution step.

eUnderlined and bold text indicates parameter set 22 is the optimized parameter sets for the single tube extraction method.

2.9.2. Development of a 96-well alkaline differential extraction method 

The Slicprep™ 96 device was used to expand the extraction method to a 96-well format. The Slicprep™ 96 device consists of a 96-well spin basket assembly, a U-shaped collar, and a 2.2mL 96-well plate (Abgene) that allow for a 665μL volume in the base of the device without coming into contact with the spin baskets when they are held up in the spin position with the U-shaped collar [27]. Sampling was conducted by transferring the tip of the swab, including the shaft, to the appropriate well of the 96-well spin basket assembly using a sterile pipette tip to position the swab at the bottom of the spin basket assembly. The assembly was covered with a 96-well plate septa mat (Axygen, Union City, California) to protect the swabs from the inadvertent transfer of DNA while allowing the wells to breathe during the heating steps. Appropriate extraction volumes were determined by empirical testing and it was found that 700μL of fluid could be used in the initial step without exceeding the 665μL maximum volume allowed in the Slicprep devices, as the swabs retain approximately 50μL of fluid. Subsequent steps were conducted in 650μL volumes to ensure sample-to-sample contamination did not occur as a result of exceeding the maximum Slicprep volume.

Cell lysis and DNase digestion steps were conducted in Boekel Digital Dry Bath Incubators as indicated above. The increased volumes and decreased contact area between the 96-well plates and the heating block inserts, as compared to the 1.5mL tubes and heating blocks, were anticipated to result in longer incubation times to reach the same temperatures as the solutions in the 1.5mL tubes during the 2min lysis steps. This was confirmed by temperature monitoring with a thermo couple (J-KEM, St. Louis, MO)(data not shown). Therefore, a 10min pre-heating step, with the plate covered with polyolefin sealing tape (E&K Scientific), was incorporated into the 96-well method for both lysis steps. Thirty second/2500rpm centrifugation steps were carried out in Hermle Z300 centrifuges fitted with microplate rotors (LabNet, Edison, NJ). As with the single-tube method, these parameters were modified and optimized by empirical testing with replicate (duplicates or triplicates) mock SAE swabs prepared from a single semen contributor and female buccal contributor to minimize variation from the sample sources. Optimized 96-well extraction parameters, parameter sets 26 and 28, are indicated in Table 3 for samples containing less than 1μg or more than 1μg of non-sperm DNA, respectively.

Table 3. Parameters used in the development of the 96-well alkaline differential extraction method.
ABCDEFGHIJKL
Parameter setμL semenNon-sperm lysis tempa (°C/min)Room temp neutralization (min)DNase digestion 37°C (min)Sperm lysis tempb (°C/min)Sperm fraction clean-up methodLoading buffer (type/mL)Wash steps (type/mL/#steps)Elution steps (eluent/μL/#steps)Identifiler™ results
MajorcMinorc
10.195/256075/2NSXSTE−4/1.2 & NTC/0.45B5/0.05/2BE/10/2FemaleND
20.195/456075/4NSXSTE−4/1.2 & NTC/0.45B5/0.05/2BE/10/2FemaleMale
30.195/556075/5NSXSTE−4/1.2 & NTC/0.45B5/0.05/2BE/10/2FemaleMale
40.195/4560d75/4NSXSTE−4/1.2 & NTC/0.45B5/0.05/2BE/10/2MixedMixed
50.195/4560d75/4NSXSTE−4/1.2 & NTC/0.45B5/0.05/3BE/10/2MaleFemale
60.195/4590d75/4NSXSTE−4/1.2 & NTC/0.45B5/0.1/1BE/10/2MaleFemale
70.195/3590d75/4NSXSTE−4/1.2 & NTC/0.45B5/0.05/3BE/10/2MaleFemale
80.195/3590d75/4NSXSTE−4/1.2 & NTC/0.45B5/0.05/3BE/10/3MaleFemale
90.195/2590d75/4NSXSTE−4/1.2 & NTC/0.45B5/0.05/3BE/10/2MaleND
100.195/1590d75/4NSXSTE−4/1.2 & NTC/0.45B5/0.05/3BE/10/2MaleFemale
110.195/1590d75/1NSXSTE−4/1.2 & NTC/0.45B5/0.05/3BE/10/2MaleND
120.195/1590d75/1NSXSTE−4/0.65 & NTC/0.325B5/0.05/3BE/10/2MaleND
130.195/1590d75/1NSXSTE−4/1.35 & NTC/0.51B5/0.05/3BE/10/2VariedFemale
140.195/1590d75/1NSXSTE−4/1.35 & NTC/0.51B5/0.05/35mM Tris pH 9.0/10/2MaleFemale
150.195/1590d75/1NSXSTE−4/1.35 & NTC/0.51B5/0.05/35mM Tris pH 9.5/10/2MaleFemale
160.195/1590d75/1NSXSTE−4/1.35 & NTC/0.51cB5/0.05/3BE/10/2MaleND
170.195/1590d75/1NS96TTE−4/1.35 & NTC/0.51B5/0.25/2TE−4e/50/1VariedVaried
180.195/1590d75/1NS96TTE−4/1.35 & NTC/0.51B5/0.25/3TE−4e/50/1MaleND
190.195/1590d75/1NS96TTE−4/1.35 & NTC/0.51B5/0.25/2TE−4e/50/1MaleND
200.195/1590d75/1NS96TTE−4/1.35 & NTC/0.51fB5/0.25/3TE−4e/50/1MaleFemale
210.195/1590d75/1NS96TTE−4/1.35 & NTC/0.51fB5/0.5 & 0.7/2TE−4e/50/1MaleFemale
220.195/1590d75/1NS96TTE−4/0.86 & NTC/1.0B5/0.5 & 0.7/2TE−4e/50/1MaleFemale
230.195/1590d75/1NS96TTE−4/0.86 & NTC/1.0B5/0.5 & 0.7/2H2Oe/30/2MaleFemale
240.195/1590d75/1NS96TTE−4/0.86 & NTC/1.0B5/0.5 & 0.7/2H2Oe/30/2gMaleFemale
250.195/1590d75/1NS96TTE−4/0.86 & NTC/1.0B5/0.5 & 0.7/21:6 TE−4e/30/2MaleND
26k0.195/1590d75/1NS96TTE−4/0.86 & NTC/1.0B5/0.5 & 0.7/21:6 TE−4e/30/2gMaleFemale
270.195/15/5h90d75/1NS96TTE−4/0.86 & NTC/1.0B5/0.5 & 0.7/21:6 TE−4e/30/2fMaleND
28kN/AjPost-coital Swab95/15/5h90d/60i75/1NS96TTE−4/0.86 & NTC/1.0B5/0.5 & 0.7/21:6 TE−4e/30/2gMalejFemalej

aNote: All non-sperm lysis steps conducted with 0.1N NaOH.

bNote: All sperm lysis steps conducted with 1N NaOH.

cNote: Only the sperm cell fractions are being assessed for the presence of male and female alleles.

dDry bath incubator set at 44°C to achieve optimal 37°C digestion temperature.

eSolution heated to 70°C prior to use as elution buffer.

fSamples loaded at 2000rpm centrifugation speed used instead of 11,000rpm.

gExtracts heated to dryness by heating @ 90°C for ∼50min.

hSubstrates rinsed in 650μL DNase buffer following DNase digestion.

iSamples subjected to a second DNase digestion step following the initial DNase digestion.

jParameter set not tested with simulated SAE swabs as previous parameter sets were optimized to the point where few, if any, female alleles were detected in the sperm fractions from these swabs and the results reported are from the authentic 24h post-coital swab.

kUnderlined and bold text indicates parameter sets 26 and 28 are the optimized parameter sets for the 96-well method with less than 1μg or more than 1μg of non-sperm DNA, respectively.

2.9.3. Verification of the absence of DNase activity in the sperm fraction extracts 

The possibility of residual DNase activity in the sperm fraction extracts was tested by processing sterile cotton swabs (2) as DNase activity check samples and a reagent blank, with the optimized single-tube and 96-well alkaline differential extraction methods, with a normalized final volume of 8μL after TE−4 buffer addition. Additionally, a tube containing 8μL of TE−4 was prepared for use as a control for comparison purposes. 1μL of 10× DNase buffer was added to each sample and control, and with the exception of the reagent blank, each was spiked with 1μL of the 32ng/μL male genomic DNA used to prepare the qPCR standards. The samples were then placed in a 37°C dry heat incubator for 1h, and a 2μL aliquot of each sample was tested with an in-house developed qPCR assay that measures the extent of DNA degradation [24].

2.9.4. Extraction of previously extracted mock sexual assault swabs 

The ability of the alkaline differential extraction to lyse spermatozoa that remain on a substrate after a standard DTT extraction was tested using swabs that had been prepared as described in Section 2.2, and extracted as described in Section 2.6, as previous work by Voorhees et al. [13] and Norris et al. [14] indicates the DTT based differential extraction is less than 100% effective at removing the spermatozoa bound to the substrate. Initially, non-sperm lysates were generated by heating the previously extracted substrate in 400μL of 0.1N NaOH for 2min at 95°C, followed by neutralization in 400μL DNase buffer and DNase I digestion in 400μL DNase I to remove residual non-sperm DNA from the substrate. Sperm fraction lysates were generated by heating the substrate in 400μL of 1N NaOH for 10min at 75°C, which were then concentrated and purified using Centricon 100s. Subsequent re-extraction experiments were conducted by replacing the 0.1N NaOH non-sperm lysis step with one or two in 400TE−4μL buffer rinses with subsequent centrifugation using Spin-X inserts (Corning) to remove the residual SEB/ProK before proceeding to the DNase buffer rinse and 1N NaOH sperm cell lysis steps. The extracts were quantified by qPCR and amplified with the Identifiler kit as described in Sections 2.7, 2.8, respectively.

2.9.5. Generation of standard differential extraction data for comparison purposes 

Mock sexual assault swabs containing 1, 0.1 and 0.01μL semen were extracted in replicate (duplicate) using the standard DTT differential extraction method described by Gill et al. [7] using Centricon 100 microdialysis units for concentration and clean-up of the sperm and non-sperm fractions as indicated in Section 2.6. Amplification of up to 10μL or 1ng of template DNA of the sperm and non-sperm fractions was conducted as indicated in Section 2.8.

2.9.6. Extraction of post-coital swabs 

Post-coital vaginal swabs taken at intervals ranging from 0 to 96h post-coitus were extracted using parameter sets 21, 26–28 (Table 3). The non-sperm fraction and cleaned up sperm fraction lysates were quantified by qPCR and typed with the Identifiler kit as indicated in Sections 2.7, 2.8, respectively.

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3. Results and discussion 

3.1. Determination of initial lysis conditions for lysis of epithelial cells in the presence of semen, spermatozoa and the development of a single-tube (1.5mL) alkaline differential extraction 

The results of the microscopic examination of the slides prepared from the epithelial cell/semen mixtures following treatment with various concentrations of sodium hydroxide at 75°C and 95°C are shown in Table 1. Based on the results in Table 1, 75°C lysis of the epithelial cells in 0.4N NaOH and 95°C lysis of the epithelial cells in 0.1N were deemed appropriate for use as initial lysis conditions for lysis of epithelial cells in the presence of semen as these lysis conditions significantly reduced the quantity of intact epithelial cells without significantly reducing the quantity of spermatozoa.

The results of the microscopic examination of the slides prepared from the rinsed spermatozoa samples are also shown in Table 1. Although 0.4N NaOH appeared to be sufficient to lyse the spermatozoa in this experiment, the presence of a single spermatozoa in the slide prepared from the 0.8N sperm/e-cell lysate, and the presence of multiple spermatozoa at 0.4N NaOH in the previous experiments, indicated that use of the 1N NaOH would be best to ensure complete lysis. Therefore, lysis in 1N NaOH for 5min at 75°C was deemed to be an appropriate for use as initial lysis conditions for the sperm-cell lysis step.

The results from initial testing of a three-step alkaline differential extraction revealed the presence of female alleles in the sperm fractions from all of the mock SAE swabs (Column L of Table 2). Subsequently, non-sperm and sperm-cell lysis conditions were varied to minimize the carryover of non-sperm DNA into the sperm fraction. In addition, a neutralization step prior to the DNase digestion step was incorporated to reduce the carryover of non-sperm DNA into the sperm fraction. STR results obtained using DNA extracted with the optimized single-tube conditions (parameter set 22) are shown in Fig. 2a.

  • View full-size image.
  • View full-size image.
  • Fig. 2. 

    (a) AmpFlSTR® Identifiler™ STR profile from sperm fraction of buccal swab spiked with 1/10μL semen that was extracted with parameter set 22 of the single-tube alkaline differential extraction method. Note: The minor alleles designated with arrows are consistent with the female “victim” type. (b) AmpFlSTR® Identifiler™ STR profile from standard organic/DTT extracted sperm fraction of buccal swab spiked with 1/10μL semen. Note: The minor alleles designated with arrows are consistent with the female “victim” type. (c) AmpFlSTR® Identifiler™ STR profile from sperm fraction of buccal swab spiked with 1/10μL semen that was extracted with parameter set 26 of the 96-well alkaline differential extraction method. Note: The minor 15 allele designated with an arrow is consistent with the female “victim” 13,15 D8S1179 type. (d) AmpFlSTR® Identifiler™ STR profile from sperm fraction of buccal swab spiked with 1/10μL semen that was extracted with parameter set 27 of the 96-well alkaline differential extraction method.

3.2. Development of the 96-well alkaline differential extraction method 

Preliminary results of extraction in the 96-well plate format yielded less than optimum sperm DNA yield and/or sperm to non-sperm DNA ratio, requiring changes in lysis times, and DNase digestion time, relative to the optimal single tube parameters. In addition, an optional second DNase digestion and post-digestion buffer rinse steps for samples expected to contain excessive quantities of non-sperm DNA were incorporated. Loading, wash and elution conditions were optimized with the NucleoSpin 96 Tissue Plates (Table 3). The final sets of optimized 96-well parameters are listed as parameter sets 26 and 28 in Table 3 and the STR results obtained from parameter set 26 are shown in Fig. 2c. Finally, it should be noted that parameter set 27, which included a post-digestion buffer rinse, resulted in reduced carry-over of non-sperm DNA into the sperm fraction to the extent that no female alleles were detected in the sperm fraction with simulated SAE swabs (Fig. 2d). However, this parameter set did not yield similar results with post-coital swabs that contained significantly higher quantities of non-sperm DNA.

Collectively, the STR results shown in Fig. 2 and the total yields of male DNA from sperm fractions indicate the alkaline differential extraction is capable of producing similar results to the standard differential extraction method. Specifically, full male profiles were yielded from mock sexual assault swabs containing as little as 0.1μL semen with few, if any, female alleles detected. Comparison of total male DNA recovered/standard deviation values for the standard organic extraction, single-tube alkaline extraction and 96-well alkaline extraction methods were 0.5ng/0.11, 0.2ng/0.03 and 0.7ng/0.08, respectively. While the yields for the different methods indicate similar recoveries, the reader is cautioned that qPCR assay variability at these low levels can be high, and we believe the STR typing results are a better practical assessment of DNA recovery.

3.3. Verification of the absence of DNase activity in the sperm fraction extracts 

Potential residual DNase activity was tested for using a multiplex qPCR assay that contains two targets of differing length. An estimate of the extent of DNA degradation is determined as a ratio of the quantity of DNA measured with each target. Theoretically, an undegraded sample will have a ratio (of short to long targets) of one, because target length is not a variable in the amplification (the sample DNA is intact), and each target will measure the same quantity. The qPCR degradation ratios for all the samples tested by spiking with DNA and incubation in DNase buffer were approximately one as shown in Table 4. Therefore there is no indication of degradation in these samples, which indicates the DNase is inactivated by heating in 1N NaOH.

Table 4. Summary of nuTH01 and nuCSF qPCR results for DNase activity check samples and DNase I degradation data.
SampleSample typenuTH01 quantity (ng/μL)nuCSF quantity (ng/μL)qPCR degradation ratio (nuCSF qty/nuTH01 qty)
S C1Single tube DNase activity check8.907.700.9
S C2Single tube DNase activity check9.209.001.0
S PosSingle tube control5.105.901.20
S NegSingle tube negative control/reagent blankNDNDN/A
96 C196-Well DNase activity check7.405.500.7
96 C296-Well DNase activity check8.005.600.7
96 Pos96-Well control6.405.100.8
96 Neg96-Well negative control/reagent blankNDNDN/A

D 0aMale DNA with 0min of DNase treatment3.603.901.1
D 15aMale DNA with 15min of DNase treatment2.803.551.3
D 17aMale DNA with 15min of DNase treatment0.901.601.8
D 27aMale DNA with 27min of DNase treatment0.551.653.0
D 42aMale DNA with 42min of DNase treatment0.351.604.5
D 62aMale DNA with 62min of DNase treatment0.251.606.4
D 92aMale DNA with 92min of DNase treatment0.131.007.7

aMean values from DNase I degradation of male (TaqMan/Raji) DNA used in nuTH01–nuSRY–nuCSF–IPC quadruplex qPCR developmental validation provided for comparison purposes.

3.4. Extraction of previously extracted mock sexual assault swabs 

Simple re-extraction of the previously extracted substrates with the alkaline differential extraction method (Table 2, parameter set 18) yielded sperm fractions that contained an average of 23.5ng of male DNA. By comparison, the original DTT differential extraction sperm fractions contained an average of 52.5ng of male DNA. The sperm fraction extracts, however, were found to be roughly equal mixtures of male and female DNA, and the non-sperm fractions completely inhibited the qPCR internal positive control (IPC). Modification of the alkaline differential extraction procedure to include one or two TE−4 buffer rinses in place of the 0.1N NaOH non-sperm lysis step, included in parameter set 18 in Table 2, yielded major male profiles (Fig. 3a and b) and a total of 30ng and 53ng of male DNA in the sperm fractions, respectively. These results indicate that less than 50% of the spermatozoa are removed from the substrate with the standard DTT differential extraction, and that these residual spermatozoa can be successfully recovered and extracted with the alkaline lysis differential extraction method, using one or more TE−4 buffer rinses as a means of removing residual sodium dodecyl sulfate (SDS), which is a known inhibitor and can significantly inhibit DNase activity at a 0.003% concentration [28]. Additionally, these results indicate that the residual SDS from the original DTT differential extraction can inhibit the DNase I as the number or female alleles detected in the sperm fractions decreased significantly with the addition of a second TE−4 buffer rinse. Two or more TE−4 buffer rinses are recommended as the concentration of SDS in our extraction buffer is approximately 600 times greater than needed to inhibit the DNase.

  • View full-size image.
  • Fig. 3. 

    (a) AmpFlSTR® Identifiler™ STRs from sperm fraction of mock SAE swab containing 1μL semen that was re-extracted with one TE−4 buffer rinse in place of the 0.1N NaOH non-sperm lysis step in the single-tube alkaline differential extraction (parameter set 18) after it had been processed with the SEB/ProK steps utilized in the DTT differential extraction. Note: The minor alleles designated with arrows are consistent with the female “victim” type and the 15 allele at D8S1179 appears to be elevated stutter at 10.5% of the 16 allele peak height. (b) AmpFlSTR® Identifiler™ STRs from sperm fraction of mock SAE swab containing 1μL semen that was re-extracted with two TE−4 buffer rinses in place of the 0.1N NaOH non-sperm lysis step in the single-tube alkaline differential extraction (parameter set 18) after it had been processed with the SEB/ProK steps utilized in the DTT differential extraction. Note: The minor alleles designated with arrows are consistent with the female “victim” type.

3.5. Standard differential extraction data for comparison purposes 

The standard differential extraction of the mock sexual assault swabs yielded full/major male profiles for swabs spiked with as little as 0.1μL semen with few minor alleles consistent with the female buccal contributor as shown in Fig. 2b. Conversely, the swabs spiked with 0.01μL semen yielded major female profiles with few, if any, male alleles detected (data not shown). Therefore, generating full profiles from mock sexual assault swabs spiked with 0.1μL semen was set as a goal for the alkaline differential extraction method prior to testing with authentic post-coital swabs.

3.6. Extraction of post-coital swabs 

The quantification results from the quadruplex qPCR assay for each of the post-coital swabs extracted with parameter set 28 are listed in Table 5, and the STR results for the sperm and non-sperm fractions from the 24h post-coital swab are shown in Fig. 4a and b, respectively. It should be noted that the 24h interval was intentionally selected as full profiles are not always generated with a standard organic differential extraction from swabs taken at this interval by analysts in training in our laboratory. While the full male profile in Fig. 4a was generated using a second DNase digestion and post-digestion rinse step, full male profiles have been generated from post-coital swabs taken 24 and 36h post-coitus without the use of a second DNase digestion or post-digestion rinse step using 96-well parameter sets 21 and 26, respectively. However, these samples contained quantities of non-sperm DNA significantly greater than ∼1μg quantities present in the mock sexual assault swabs (Fig. 2a and c) and as a result numerous female alleles were also detected in these samples (data not shown). The second DNase digestion and post-digestion rinse steps may be considered optional as they are not required for samples expected to contain quantities of non-sperm DNA less than 1μg (i.e. as indicated by microscopy during screening when the total number of epithelial cells are estimated to be less than approximately 170,000). Finally, the ability to detect male DNA in sperm fractions from post-coital swabs collected up to 96h post-coitus (Table 5) with the 28 cycle Identifiler kit suggests that collection of sexual assault evidence out to or beyond 96h from the time of the sexual assault is prudent as more sensitive typing systems (i.e. Identifiler Plus, PowerPlex 16, PowerPlex 16 HS, etc.) that incorporate enhanced amplification chemistries and additional PCR cycles are likely to yield more complete male profiles.

Table 5. Summary of nuTH01–nuCSF–IPC qPCR quantification and AmpFlSTR® Identifiler™ STR results for post-coital swabs extracted with parameter set 28.
Sample no.Sample typeAdjusteda extract volume (μL)nuTH01 quantity (ng/μL)Total DNA recovered (ng)Identifiler™ amelogenin X/Y peak heights (RFU)Identifiler™ % male/%female alleles detectedb
0 SSperm fraction – post-coital swab (0h interval)124.70056.402966/3085100/0
24 SSperm fraction – post-coital swab (24h interval)120.0240.29636/601100/4
48 SSperm fraction – post-coital swab (48h interval)120.0050.06158/6220c/22c
72 SSperm fraction – post-coital swab (72h interval)120.0040.05340/ND3d/63
96 SSperm fraction – post-coital swab (96h interval)120.0200.25539/590d/80
RB SSperm fraction – reagent blank12NDNDNDN/A
0 NSNon-sperm fraction – post-coital swab (0h interval)6914.60030311560/17597/100
24 NSNon-sperm fraction – post-coital swab (24h interval)69110.50069191872/ND0/100
48 NSNon-sperm fraction – post-coital swab (48h interval)69113.00085671765/ND0/100
72 NSNon-sperm fraction – post-coital swab (72h interval)69122.00014,9981912/ND0/100
96 NSNon-sperm fraction – post-coital swab (96h interval)69129.00019,1112310/ND0/100
RB NSNon-sperm fraction – reagent blank691NDNDNDN/A

aVolume of extract after the sperm fractions were reconstituted in 12μL H2O or the non-sperm fractions were neutralized with 59μL 2M Tris pH 7.5.

bNote: PCR volume for this amplification set was inadvertently increased to 28μL that consisted of 18μL of Identifiler™ master mix and 10μL extract, but did not yield any notable difference in the 9947A positive control data as compared to previous 25μL 9947A data.

cIncluding overlapping alleles that may have been contributed by the male or female as the amelogenin data suggests a near equivalent mixture of male and female DNA.

dExcluding overlapping alleles as the amelogenin data suggests the male component is the minor component.

  • View full-size image.
  • Fig. 4. 

    (a) AmpFlSTR® Identifiler™ STRs from sperm fraction of vaginal swab taken 24h post-coitus that was extracted with parameter set 28 of the 96-well NaOH–DNase differential extraction method and typed within 24h from time of sampling. Note: The minor 16 allele designated with an arrow is consistent with the female “victim” 16,16 D19S433 type. (b) AmpFlSTR® Identifiler™ STRs from non-sperm fraction of vaginal swab taken 24h post-coitus that was extracted with parameter set 28 of the 96-well NaOH–DNase differential extraction method and typed within 24h from time of sampling.

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4. Conclusions 

A differential extraction method that allows up to 96 sexual assault evidence samples to be simultaneously extracted within a few hours could greatly improve throughput and productivity for laboratories experiencing sexual assault evidence sample backlogs. The reduction of these backlogs could go a long way to help solve numerous suspectless sexual assaults through the use of national databases like CODIS, and to prevent future crimes with the identification of serial offenders. Therefore the described alkaline differential extraction method was intentionally developed with inexpensive reagents and designed with a minimum number of steps. The absence of repeated handling steps both minimizes the possibility of contamination and greatly reduces the demand on laboratory staff. In our hands this procedure has reproducibly yielded major male STR profiles in less than 24h from the time of sampling, with simulated sexual assault swabs containing as little as one-tenth of a microliter of semen and with post-coital swabs taken up to 24h post-coitus. The procedure was developed for use with cotton tip swabs that are generally used in the collection of sexual assault evidence in the State of California, but has also been successfully tested with a wide variety of swabs, including polyester tipped swabs that are sometimes used in the collection of sexual assault evidence. It should be noted however that the recoveries of DNA from the cotton tipped swabs tended to be slightly higher than those from polyester swabs (data not shown), which is likely the result of the spermatozoa being better retained during the extraction process by the cotton as suggested by Benschop et al. [29]. Additionally, while the alkaline differential extraction method was developed with mock swabs prepared from semen and buccal swabs from one set of contributors to minimize sample-to-sample variation, it has since yielded similar results from mock swabs prepared from two additional semen contributors and five additional buccal contributors (data not shown). However, the alkaline differential extraction method has not been tested with sexual assault evidence swabs that have been stored under conditions other than frozen storage, and the authors would strongly suggest that laboratories with these types of samples conduct stability studies prior to utilization of this method.

As pointed out, it has been suggested that the standard DTT based differential extraction is less than 100% effective at the removal of sperm from a cotton fiber swab. Our results with re-extraction using the alkaline lysis procedure, suggest that less than 50% of the male DNA present on a swab is captured with a standard DTT extraction method. In cases where minimal or borderline amounts of DNA are recovered, this limitation can mean the difference between a successful typing result, and no or only partial results being obtained. Swabs previously extracted with a standard DTT differential protocol can be expected to still contain male DNA, and in cases where recovery was less than optimal it is possible to retrieve more DNA with the alkaline procedure described here.

The California Department of Justice administers a unique program, called Fast Track Forensics that requires immediate submission of sexual assault evidence directly from the point of collection to the laboratory, and the completion of a CODIS search within five working days of receipt of the evidence. The short turnaround time requirement for these cases necessitates optimal efficiency at every step. The alkaline differential extraction procedure described here allows for the simultaneous DNA extraction of up to 96 sexual assault evidence items in approximately 4h, without the use of a robotic system and with a minimum of sample handling. Results with this procedure, in terms of the successful generation of an uploadable STR profile, are about the same as with the standard DTT organic extraction. Its rapid turnaround time, ease of use, and relatively inexpensive cost to implement makes it a viable alternative to the standard organic DTT differential DNA extraction method currently used in most forensic laboratories, especially those with high throughput requirements.

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Acknowledgements 

The authors thank Andrea Bramwell and Brian Ruf (E&K Scientific), Markus Meusel and Marie Harnetiaux (Macherey-Nagel), Chris Collopy and Susan Shrader (Puritan Medical Products), Douglas Kraus (ThermoFisher Scientific), Abel Guiterez (V&P Scientific) and Carrol Brown, Mar Lin Anderson, Melinda Gee and Sheila Frieson (CA DoJ Jan Bashinski DNA Laboratory Purchasing Group) for the assistance in obtaining each of the items used in the development of the alkaline differential extraction method. Additional thanks to the laboratory staff who provided the various swabs and biological fluids used in the development of the alkaline differential extraction method and to Mavis Date Chong (CA DoJ Jan Bashinski DNA Laboratory) for sharing the details of her work on the CALDoJ Databank sodium hydroxide extraction method and the DNase digestion of samples for our quadruplex qPCR assay validation, which were ultimately merged into the alkaline differential extraction method. Finally, a special thanks to the anonymous reviewers for their thorough review of this manuscript.

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PII: S1872-4973(11)00013-5

doi:10.1016/j.fsigen.2010.12.015

Forensic Science International: Genetics
Volume 6, Issue 1 , Pages 1-16, January 2012

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