Rapid Processing of Sexual Assault Samples Utilizing the Casework Direct Kit and a Customer Developed Differential Lysis Protocol
David Carrick-White, Promega
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Prior to the introduction of the Casework Direct System (Cat.# DC4560, DC4561, Promega), the product was available to customers as the Casework Direct Kit, Custom (Cat.# AX4560, Promega). This kit has been tested by multiple forensic DNA laboratories to expedite the processing of common casework sample types, such as blood and saliva on various matrices, cigarettes, semen and trace DNA on swabs and tape.
The Casework Direct System protocol entails a simple and rapid lysis of the sample, requiring no tube-to-tube transfers or DNA binding steps, producing a lysate compatible with RT-PCR quantification and PowerPlex STR amplification systems.
The Casework Direct System kit includes a reducing agent, 1-thioglycerol, and is capable of lysing sperm in the standard protocol. The protocol can be optimised in customer laboratories and Kharlamov et al produced a protocol variant for a differential lysis method utilizing the Casework Direct System kit to expedite the process. This allows sexual assault samples that are a mixture of sperm and epithelial cells to be more rapidly processed using the Casework Direct System kit, resulting in a sperm fraction containing the spermatozoa from the seminal fluid and an epithelial fraction derived from non-sperm cells present in the sample. The DNA from these two fractions can be quantified and amplified with Promega Systems without further purification.
This modified protocol was shared with Promega and a small-scale test, detailed below, was conducted.
5ul of neat semen was pipetted onto 4 buccal swabs taken from a female donor. The swab heads were sampled into 1.5ml snap lock tubes. The four tubes were processed with the Casework Direct kit in the following manner:
Epithelial cell lysis: 200ul of Casework Direct reagent (without 1-thioglycerol) was added to the tube and incubated at 56°C for 15 mins on a thermomixer at 1000rpm. The swab head and lysate were transferred to a DNA IQ™ spin basket (Cat.# V1225, Promega) previously assembled into a labelled ClickFit Microtube, 1.5ml (Cat.# V4745, Promega). This assembly was centrifuged at maximum speed (14,000rpm) for 5 mins, after which the spin basket containing the swab was removed and discarded. 100ul of supernatant was carefully transferred to a new 1.5ml tube. This epithelial DNA fraction is ready for quantification and subsequent STR analysis.
Sperm pellet washing: The remaining supernatant was removed, avoiding the tube walls where the seminal pellet may have formed, and discarded. The sperm pellet was washed by the addition of 200ul of Casework Direct reagent (without 1-thioglycerol). The tube was vortexed at high speed for 15 seconds and centrifuged at 14,000rpm for 5 minutes. The supernatant was removed and discarded, leaving approximately 20ul. This wash step was repeated once more with Casework Direct reagent and twice more with 200ul of Water, Amplification Grade.
Sperm cell lysis: 50ul of Casework Direct reagent (including 1-thioglycerol) was added to the sperm pellet. The tube was vortexed for 15 seconds and incubated on a thermomixer (70°C for 30 minutes at 1000rpm) to lyse the sperm cells.
Both the seminal and epithelial cells fractions were quantified with the PowerQuant System (Cat.# PQ5002, PQ5008, Promega), following the procedure in the Technical manual (TMD047 Rev. 1/20) and using the Applied Biosystems® 7500 Real-Time PCR System. The quantification data was processed with the PowerQuant Analysis Tool.
All samples were amplified using the PowerPlex Fusion 6C system (Cat. # DC2705, Promega) optimised for a 1ng input at 29 cycles. DNA fragment analysis was carried out on an Applied Biosystems® 3500xL Genetic Analyzer and the data was analysed with GeneMapper ID-X® Software v. 1.5 (Thermo Fisher).
The PowerQuant quantification results can be seen presented in figure 1 and reported in chart 1.
The 4 seminal fraction samples produced similar autosomal concentrations with a mean of 5.7 ± 0.31 ng/ul. The ratio of autosomal DNA to male DNA ([Auto]/[Y]) was <1.36 for all 4 samples suggesting the samples consisted of a mainly male derived DNA contribution. The ratio of the lower molecular weight autosomal target compared to the higher molecular weight autosomal target ([Auto]/[Degradation]) was <1.35 for all 4 samples, suggesting no degradation of the DNA. The Internal Positive Control (IPC) amplified as expected in all 4 samples, indicating that no amplification inhibitors were present. These results suggested that the seminal fraction samples should amplify to produce STR profiles of good quality with good intra-colour balance.
The 4 epithelial fraction samples produced autosomal concentrations with a mean of 2.8 ± 0.58 ng/ul. The ratio of autosomal DNA to male DNA ([Auto]/[Y]) was between 7.1 and 15.6 for all 4 samples. This suggested the samples consisted primarily of DNA derived from a female contribution, with a minor contribution derived from a male donor(s). The [Auto]/[Degradation] ratio of between 1.49 and 2.9 for the epithelial samples was slightly higher than the seminal fractions. These ratios would not be expected to correlate to a noticeable imbalance or dropping out of the higher molecular weight fragments in the STR profile. The IPC showed a slightly delayed amplification of <0.3 cycles in 3 of the 4 samples, however, this deviation was not sufficient to expect the resultant STR profiles to manifest signs of PCR inhibition. The other sample showed a more significant deviation (IPC shift of 0.77 cycles) and suggested the possibility of a sub-optimal STR result owing to a level of inhibition.
Figure 1. PowerQuant sample DNA concentrations
Chart 1. Sample quantification data - PowerQuant Analysis Tool
The seminal fraction samples produced similar STR results with mean profile peak heights of 3428 ± 733 RFU. All results were full, single source male profiles of good quality. An example is presented in figure 2.
Figure 2. PowerPlex Fusion 6C seminal fraction sample 1 electropherogram
The 4 epithelial fraction samples also produced similar PowerPlex Fusion 6C results. All 4 results were mixed profiles consisting of a major female contributor and a lower level male contribution. An example is presented in figure 3. The female profile was a full profile in all 4 samples, although there was some evidence of a slight decline in peak height towards the higher molecular weight fragments. The minor contribution was consistent with the seminal donor. The single, low level alleles present at each of the Y-STR loci support a male contribution from a single donor.
Figure 3. PowerPlex Fusion 6C epithelial fraction sample 1 electropherogram
The results show that the Casework Direct reagent can be utilized without 1-thioglycerol to achieve lysis conditions suitable for lysing epithelial cells, while preserving a population of intact sperm cells. This reagent could also be used to wash these sperm cells without eliminating them.
The epithelial fraction samples all contained a minor contribution consistent with the seminal donor. It is unclear if this is due to the premature lysis of a small proportion of the sperm cells, or if this DNA is derived from male epithelial cells present in the semen sample, or a contribution from both.
The epithelial fraction STR profiles also contain a minor reduction in the higher molecular weight peak heights. This could be due DNA degradation inherent in the sample or it may be due to decreased efficiency in the PCR reaction for the epithelial samples. This may be supported by the slight delay in the PowerQuant reaction IPC of these samples compared to the seminal samples. It should be noted that Casework Direct lysates are processed with a 70°C incubation for 30 minutes in the standard protocol and that this is considered to be a requirement for PCR compatibility. This step is not included in this modified protocol and the bioactivity of the reagents may not have been completely inactivated in the epithelial fraction.
The data confirmed that in the presence of 1-thioglycerol, the Casework Direct reagent efficiently lyses sperm cells to produce a lysate that is fully compatible with downstream PowerQuant and PowerPlex Fusion 6C processing.
The lysates produced from the seminal fractions had higher concentrations of DNA than the epithelial fraction samples. When considering DNA recovery, it is important to consider the differing final lysate volumes of the seminal and epithelial fractions in this method. The seminal fractions were lysed in 50ul of Casework Direct reagent and therefore contained a mean of 285ng of DNA. The epithelial fractions were lysed in 200ul and contained a mean of 560ng of DNA.
The proposed customer protocol functioned successfully as a differential protocol to process mixtures of semen and epithelial cells, creating lysates that were compatible with Promega quantification and PowerPlex amplification systems. The [Auto/Y] ratios reported by PowerQuant were good predictors of the relative profile contributors. Further optimisation of the epithelial lysis step should be considered to enhance downstream compatibility when processing samples with low level epithelial contribution, where the epithelial STR result is important.
The lysis steps in the method are comparatively short compared to many traditional methods and the absence of subsequent purification steps shortens the length of time required to process this sample type. Additionally, the absence of these additional steps and the use of reagents manufactured in alignment with the ISO 18385 standard minimises the risk of human DNA contamination.
More extensive testing and further optimisation has been carried out and is continuing in customer laboratories. This includes data generated from a variety of real casework samples.
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