STR Analysis to Determine a Match Between Two DNA Samples

2.1 DNA Transfer

DNA can be transferred through three main mechanisms: direct transfer, secondary transfer, and tertiary transfer. Direct transfer involves the direct transfer of DNA from one item to another. Secondary transfer occurs when DNA is deposited onto an object through an intermediary, while tertiary transfer involves the passing of DNA through two intermediaries.

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Lockard's exchange principle posits that every contact leaves a trace. The type of transfer can also impact the quality of the recovered DNA. DNA subjected to tertiary transfer may result in reduced DNA quantity, as it has traversed through two intermediaries. Moreover, DNA that has undergone multiple stages of transfer can be subject to degradation.

2.2 DNA Extraction

Following DNA recovery, the next step is the extraction of DNA from the evidence sample. DNA extraction involves three key processes: extraction, purification, and isolation.

Extraction is the process by which DNA is removed from its cellular casing. Purification entails the removal of any inhibitors present, while isolation involves capturing any remaining DNA for analysis. In this study, modern silica extraction was chosen as the preferred method for DNA extraction. Proteinase K was used to digest the proteins, and Buffer ATL, containing chaotropic salt, facilitated DNA binding to the silica membrane. The samples were then incubated at 56°C to encourage further proteinase K digestion, followed by incubation at 70°C to denature the proteinase K, preventing further digestion. The primary objective of this process was to lyse the cells within the sample, eliminating unwanted cellular components and inhibitors.

2.3 DNA Quantification Using PCR

DNA quantification was performed using polymerase chain reaction (PCR) and thermal cycling. Samples BCPF-1, BCPF-2, BCPF-3, and BCPF-4 were subjected to this quantification method, which allows for the replication of DNA sequences through PCR, making it a valuable alternative to restrictive endonucleases and ligase methods (Mullis B K et al., 1994). Thermal cycling amplifies the DNA chain's size through three cycles: denaturation, annealing, and extension. It is recommended to limit the cycles to around twenty-seven to twenty-eight, as excessive cycles may lead to DNA denaturation (Watanabe, Y et al., 2003).

2.4 STR Analysis

Short Tandem Repeats (STRs) analysis is a DNA analysis method used to examine unknown DNA samples. Most individuals share common DNA regions, and STR analysis allows differentiation between DNA samples. Capillary electrophoresis is employed to detect STR alleles (Butler, J M, 2012).

3.1 Quality Control and Quality Assurance

Working with DNA in a laboratory setting necessitates rigorous steps to minimize contamination risks involving DNA samples, laboratory equipment, and workstations. Personal protective equipment (PPE), including a fully buttoned lab coat, hair net, beard guard (if required), lab glasses, and gloves, must be worn. In addition to PPE, strict procedures must be followed to prevent further contamination. Using the open-close method, where only one container is open at a time while pipetting samples, ensures minimal cross-contamination. Similarly, when replacing pipette tips, the disposal container must be closed before opening another container containing new pipette tips. Utilizing a centrifuge to separate sample contents minimizes the risk of spillage upon opening the tube. Post-experiment, thorough cleaning of all surfaces and equipment is essential to disinfect the laboratory, making it available for subsequent use. Upon exiting the laboratory, proper disposal of face coverings and gloves followed by handwashing is imperative.

3.2 Casework Samples

The collected samples included:

• BCPF-1: Cigarette from the front porch
• BCPF-2: Reference sample from the victim
• BCPF-3: Swab from the blood stain on the male suspect's shirt cuff
• BCPF-4: Reference buccal swab from the male suspect
• BCPF-5: Upper vaginal swab from the deceased woman

BCPF-1 and BCPF-3, the crime scene samples, were processed separately from BCPF-2 and BCPF-4, the reference samples. Both crime scene samples underwent extraction processes using the QIAamp DNA Investigator kit supplied by Qiagen (Qiagen, 2020), designed to recover cellular material.

3.3 DNA Extraction

For the DNA extraction of crime scene sample BCPF-1 (cigarette bud), the Qiagen QIAamp DNA Investigator kit was utilized, following the manufacturer's provided method guide (Qiagen, 2020). Similarly, for crime scene sample BCPF-3 (swab from the blood stain on the cuff), Qiagen's QIAamp DNA Investigator kit was employed, adhering to the manufacturer's published method guide (Qiagen, 2020). During the extraction process, the 007 positive control and water for the negative control (Thermofisher, 2018) were included alongside both crime scene samples.

3.4 DNA Quantification

BCPF-1 and BCPF-3 were subjected to DNA quantification via PCR, following a modified method (Walker A, J, 2003). A 1 in 5 series dilution was employed using 50 ng/µL of DNA for seven standards to determine the necessary reaction mix. The consumables utilized included 52 µL of 1X PCR buffer, 286 µL of water, 52 µL of 1 µM Alu primer forward (Alu 3- 5' GATCGCGCCACTGCACTCC 3'), 52 µL of 1 µM Alu primer reverse (Alu 5-5' GGATTACAGGTGAGCCAC 3'), and 52 µL of 1X DNA polymerase. Nineteen µL of the master mix was divided into 24 individual PCR tubes, as the experiment was conducted in duplicate, along with 1 µL of the DNA sample. The samples were centrifuged to ensure thorough mixing. Thermal cycling involved an initial denaturation step at 95°C for 2 minutes for the first cycle, followed by denaturation at 95°C for 5 seconds for 39 cycles. The annealing and extension step was set at 60°C for 40 seconds, repeated for 39 cycles. The final DNA concentrations for the samples were as follows: BCPF-1 (0.26 ng/µL), BCPF-2 (15 ng/µL), BCPF-3 (10.9 ng/µL), and BCPF-4 (21 ng/µL) (Gill, P et al., 2000).

3.5 SGMPLUS Profile Setup

SGMPLUS profile setup was carried out using the AmpfSTR SGM Plus PCR amplification kit, following the ThermoFisher manufacturer's guide (ThermoFisher Scientific, 2018).

3.6 Analysis

After thermal cycling, appropriate thresholds for amplification were set based on the observed noise in the electropherograms of each sample. Three separate channels were designated for alleles, each with its threshold: Channel 1 threshold was set at 19.05 RFU, Channel 2 threshold at 26.82 RFU, and Channel 3 threshold at 28.90 RFU. These thresholds were established to exclude 99% of the noise produced during interpretation. The electropherogram data for samples BCPF-1 and BCPF-3 were entered into a match probability calculator. The formula for calculating match probability depended on whether the locus was heterozygous or homozygous.

For heterozygous loci, the formula used was:

2⋅(((G₉)⋅(1−J₉)⋅J₉)⋅(G₁₀⋅(1−J₉)⋅J₉))⋅((1−J₉)⋅1⋅(2⋅J₉)).

For homozygous loci, the formula used was:

((G₇⋅(1−J₉))⋅(2⋅J₉))⋅(G₇⋅(1−J₉)⋅(3⋅J₉))⋅((1−J₉)⋅(1⋅(2⋅J₉))).

4.1 Electropherograms and Sample Analysis

All electropherograms provided the necessary information with no more than two peaks observed at any given locus when appropriate thresholds were applied, indicating the absence of sample mixing. The electropherogram of the negative control demonstrated no amplification, confirming the correct execution of the negative control procedure. The STR table reveals that sample BCPF-1 exhibited DNA profiles only for the loci D3S1358, AMEL, D8S1179, and FGA. Consequently, the remaining locus with no STR profile data will be excluded from further analysis due to insufficient data.

A) Electropherogram for BCPF-2 (Victim's Reference Sample)

The electropherogram labeled A above displays the DNA profile of the crime scene sample BCPF-2, taken from the victim, in three separate channels. All heterozygous loci exhibited balanced percentages of 85%, while the locus FGA displayed a stutter percentage of 20%, exceeding the laboratory acceptance threshold by 5%.

B) Electropherogram for BCPF-3 (Blood Stain on Cuff)

The electropherogram labeled B above represents the DNA profile of crime scene sample BCPF-3, derived from the blood stain on the cuff. Only the locus D195433 displayed an 85% heterozygous balance, with the remaining heterozygous balances falling below 85%, except for vWA, which exhibited a balance of 93%. The locus FGA displayed a heterozygous balance of 43% with a 20% stutter (Kelly, H et al., 2012).

C) STR Profile Table for Samples BCPF-1, BCPF-2, BCPF-3, BCPF-4

The STR profile table labeled C above presents the DNA profiles of samples BCPF-1, BCPF-2, BCPF-3, and BCPF-4, along with the 007 positive control and the corresponding loci. BCPF-1 yielded only four visual STR profiles (Alaeddini, R., 2012). Both BCPF-2 and BCPF-3 provided sufficient DNA, revealing identical STR profiles, including the AMEL locus that determines the sex chromosome. BCPF-4 also yielded enough DNA, allowing for a potential match between the loci identified in sample BCPF-1 and those in sample BCPF-4.

D) Match Probability Calculator for BCPF-1 (Cigarette from Front Porch)

The match probability calculator labeled D above displays the combined match probability and likelihood ratio for DNA crime scene sample BCPF-1, the cigarette from the front porch. Allele frequencies within the electropherogram channels were inputted alongside the respective loci, generating three individual locus-specific match probabilities. Based on the STR typing results of sample BCPF-1, it is not sufficient to conclusively exclude the possibility that the biological material originates from the suspect. However, the likelihood of an individual having three amplified loci matching those from the BCPF-1 sample is calculated to be 1 in 5,268.57492 based on the population allele frequency database.

E) Match Probability Calculator for BCPF-3 (Blood Stain on Cuff)

The match probability calculator labeled E above presents the combined match probability and likelihood ratio for DNA crime scene sample BCPF-3, the blood stain found on the cuff. Allele frequencies for each locus were entered. The individual allele frequencies yielded specific locus match probabilities. Based on the STR typing results of sample BCPF-3, it can be asserted with a high degree of confidence that the blood sample belongs to the victim. The probability of an unrelated individual possessing the same STR profile matching the DNA obtained from BCPF-3 is calculated to be 1 in 240,402,972,124,104.00000 based on the population allele frequency database.

5.1 Analysis of BCPF-1 (Cigarette from Front Porch)

As mentioned in the results section, DNA crime scene sample BCPF-1 (cigarette found on the porch) yielded only four loci within the STR profile, rendering the remaining loci unsuitable for interpretation. Various factors may account for the insufficient DNA recovery, including the presence of inhibitors (Moore, D., 2019). Environmental factors, such as weather conditions, could have impacted the sample's integrity, leading to degradation and hindering DNA recovery. Another contributing factor could be the type of DNA, as saliva contains significantly less DNA (5000 ng/mL) compared to samples like blood and semen (Schmerer, WM, et al., 1999). Based on the results, it can be inferred that the likelihood of another individual having three amplified loci matching those from BCPF-1 is 1 in 5,268.57492. The data suggests that the sample BCPF-1 is most likely linked to the suspect, as the higher the likelihood ratio, the fewer alleles are amplified, allowing for a broader population match. The four visible loci in BCPF-1's STR profile match identically with the loci of BCPF-4 (the buccal swab). For instance, the AMEL locus, which denotes the male XY chromosome, is consistent between both samples, reinforcing the likelihood that sample BCPF-1 belongs to the suspect, as confirmed by the reference sample BCPF-4.

5.2 Analysis of BCPF-2 (Reference Sample from Victim)

DNA sample BCPF-2, the reference sample from the victim, displayed an 85% heterozygote balance, indicating proper processing without interference. However, the locus FGA exhibited a 20% stutter, surpassing the laboratory threshold by 5%. The likely causes for this discrepancy could include allelic drop-in (Moore, D., 2020) due to a degraded sample or lapses in laboratory practice. To prevent this, improvements in laboratory techniques, such as pipetting skills and maintaining a sanitized work environment, should be implemented. It can also be noted that the AMEL locus in sample BCPF-2 displayed the expected female XX chromosome. This supports the integrity of the sample. Overall, BCPF-2's DNA profile appears consistent with that of the victim, considering the XX chromosome.

5.3 Analysis of BCPF-3 (Blood Stain on Cuff)

DNA sample BCPF-3, obtained from the blood stain on the cuff, displayed two loci of interest. The locus FGA exhibited a heterozygote balance of 43%, potentially indicating issues such as degraded DNA or poor laboratory practice (Hunt, J., 2009). The suspect's statement mentioned a recent shaving cut, which could explain the suboptimal heterozygote balance in the FGA locus if the blood belonged to the victim. The 20% stutter observed in the FGA locus could account for the 43% heterozygote balance, although poor laboratory practice should also be considered. Sample BCPF-3 also revealed the AMEL locus with an XX female chromosome, contradicting the suspect's claim that the blood was his (which would have displayed an XY chromosome). As a result, it is highly likely that crime scene sample BCPF-3 belongs to the victim.

6.0 Conclusion

Based on the information presented in the results and discussion sections, it can be reasonably concluded that samples BCPF-2 and BCPF-3 are highly likely to be a match. Both samples share the same STR profile and exhibit matching chromosomal sexes. The investigation process did not consider the possibility of bad laboratory practices, which could have affected the results if incorrect procedures were followed, such as inadequate pipetting skills, equipment misuse, or improper use of personal protective equipment (PPE). Additionally, the process may not have accounted for the challenges posed by degraded samples, which can yield less interpretable results, rendering them ineffective for the investigation. To address these issues, preventive measures against bad laboratory practices should be enforced, ensuring that all participants are aware of the expected standards when handling DNA samples. For degraded samples that prove challenging to interpret, alternative methods such as "PCR amplification and boosted capillary electrophoresis" (Westen, A.A et al., 2009) can be employed to enhance the sample's observability and increase its value in the investigation.