Abstract
Forensic investigations using DNA analysis have been grown rapidly. Samples retrieved from crime scene may be exposed to different conditions before proceeding. This study aimed to evaluate the effect of different grades of temperature and burn on DNA extraction and typing.
Methods:
Seven mL of blood and four mL of semen were collected from each volunteer. Effects of temperature grades (100 °C, 50 °C, 37 °C, 4 °C, −20 °C, and burn) on blood and seminal stain were tested.
Results:
Bloodstains exposed to temperature grades 100 °C, 50 °C, 37 °C, 4 °C, and −20 °C can be identified using preliminary test while burnt blood stain cannot. Seminal stains exposed to temperature grades 37 °C, 4 °C, and −20 °C can be identified by Florence test while those exposed to 100 °C, 50 °C, and burn cannot. Blood and seminal stains exposed to temperature grades 100 °C, 50 °C, and burn show marked reduction in DNA concentration while maximum DNA conc could be recovered from stains exposed to temperature grade temperature. Both blood and seminal DNA was affected only in case of burn without significant difference between THO1 and Amelogenin primers.
Conclusion:
High environmental temperature affect the quantity of extracted DNA from different stains but less effect on the quality of extracted DNA. Burn affects both preliminary test, DNA quantity, and quality in stains.
Keywords: Forensic pathology, Bloodstains, Seminal stains, DNA degradation, Gel electrophoresis
Introduction
DNA fingerprinting is a technique, which helps scientists and legal experts to identify and solve crimes (1). DNA analysis in forensic medicine routinely deals with materials recovered from crime scenes, paternity testing, and identification of human remains (2). It is so sensitive test that too small sample can be used to link suspects to crime scenes. Ability to retrieve genetic information from the contents of just a few cells has made DNA profiling one of the most relied upon disciplines in any forensic department. It is the single most important event in forensic medicine in the late 20th century (3).
To be accepted in court, sample collection from crime scene for DNA analysis must be performed carefully, to produce DNA profiles. Carefully collected, preserved, stored, and transported prior to any analysis conducted in a forensic DNA laboratory (4). The conditions, which samples exposed to before proceeding, is very important. These can lead to DNA degradation like ultraviolet (UV) light, humidity, and temperature (5). Fire is frequently used by assailants to cover up homicides or other violent (6). Forensic laboratory should deal with these samples that are less than ideal (5); and (7).
Blood is a common body fluid which can be present at the crime scene (8). Seminal stains left at a crime scene also is an important evidence in many types of crime including sexual crimes (9).
This work sheds light on the effect of different grades of temperature and burn on the quantity and quality of DNA retrieved from blood and seminal stains.
Subjects, Materials, and Methods
The present study was conducted from the 1st of March 2018 to the 30th of April 2018. Samples (blood and semen) were collected from 20 male volunteers after taking informed written consent, all of them were patients in Andrology clinics, Assiut University Hospitals. Samples were analyzed in Forensic Medicine and Clinical Toxicology department, Faculty of Medicine, Assiut University, and molecular biology unit.
Inclusion Criteria
Age group was from 20 to 40 years old (10).
Exclusion Criteria
There is no any exclusion criteria as STR Short tandem repeat core loci not known to be affected by any medical conditions (11).
Sample Processing
Blood and semen samples collection
Seven mL of blood and four mL of semen were taken from the same volunteer. Blood samples were taken by venipuncture and semen samples were taken by masturbation. The blood samples were collected in two 6 mL vacutainer K2E EDTA tubes (3 mL each), and semen samples were collected in a plastic sterile container. The blood and semen samples were assigned codes to protect the privacy of the donors and allow for unbiased evaluation (12).
Blood and semen sample processing
Each blood sample was divided into seven parts, one mL each spotted on seven pieces of autoclaved white cotton fabric (3 × 3 cm) (13). The cotton fabric was previously tested for blood DNA without the addition of samples as negative control. They were left over night to dry at average ambient room temperature in Assiut governorate (18.2 °C-23 °C) (14). They were left on laboratory counter after its sterilization with ethyl alcohol (15). First sample was assigned as 1B* and served as positive control and was processed immediately after drying, the rest of the samples were classified and exposed to different grades of temperature and burn.
Each semen sample was divided into seven equal parts half mL each by dropper and was spotted on a piece of autoclaved white cotton fabric (3 × 3 cm). The cotton fabric was previously tested for seminal DNA without the addition of samples as negative control. Then the cotton fabric pieces containing the seminal stain were left over night to dry at average ambient room temperature in Assiut governorate (18.2 °C-23 °C) (14). They were left on laboratory counter after its sterilization with ethyl alcohol (16) . First sample was assigned as 1S* and served as positive control and was processed immediately after drying for 24 hours, the rest of the samples were classified and exposed to different grades of temperature and burn.
Exposure to different grades of temperature and burn
Blood samples from 1B (a) to 20B(a) and seminal samples from 1S(a) to 20S(a) were put in paper envelope and put into oven for one day where the temperature was adjusted to 100 °C pre heated to the wanted degree (Jouan oven model & serial No. 39312318) (15).
Blood samples from 1B(b) to 20B(b) and seminal samples from 1S(b) to 20S(b) were put in paper envelope and put into oven for one day where the temperature was adjusted to 50 °C (15).
Blood samples from 1B(c) to 20B(c) and seminal samples from 1S(c) to 20S(c) were put in Eppendorf tube and exposed to temperature 37 °C in incubator (Opticivymen system) for one day (17).
The samples from 1 (d) to 20 (d) either blood or semen were put in paper envelope into the refrigerator for one day where the temperature was adjusted to 4 °C (18).
The (e) samples from 1 to 20 either blood or semen were put in paper envelope into the deep freezer for one day where the temperature was adjusted to −20 °C (17).
Lastly, blood samples from 1B(k) to 20B(k) and seminal samples from 1S(k) to 20S(k) were burnt in a glass dish by matches until charring (19).
Examination of the Samples
Macroscopic examination of blood and semen samples
Each sample was examined by naked eye and photographed for the visual identification of blood and semen and their characters (20).
Preliminary tests
Each piece of cloth was divided into two equal halves. Presumptive tests: Kastle Mayer test (phenolphthalein test) for blood samples and Florence test for semen samples were done for half of the piece of cloth.
DNA analysis
The other half of the cloth was used for DNA analysis.
DNA Extraction from Blood and Seminal Stain
First, the blood and seminal stain samples were extracted from half of the piece of cloth by addition of 600 µg of lysis buffer to the piece of the cloth in Eppendorf tube, incubated at room temperature for one hour, then vortexed for 15 sec, and centrifuged at 13 000 for 1 min. (21).
Purifying genomic DNA from whole blood and semen
Blood DNA was extracted using DNA extraction kit (Bioline company, Cat No. BIO_52063) following the manufacturer protocol (22). Seminal DNA also was extracted using DNA extraction kit (Bioline company, Cat No. BIO_52066) following the manufacturer protocol (23).
DNA quantification by spectrophotometer for blood and semen samples: according to Hue et al. (21)
After extraction, Nano Drop Spectrophotometer ND-1000 estimates quantity of extracted DNA. DNA absorbs UV light in a specific pattern. In a Spectrophotometer cuvette, a five µm of the sample was exposed to UV light at 260 nm and 280 nm, and a photodetector measured the light that passes through the sample. The concentration of extracted DNA was assessed at 260 nm and 280 nm. The ratio between the reading at 260 and 280 nm (OD 260/280) provided an estimation of purity of DNA.
DNA amplification
This was done by using the polymerase chain reaction (PCR), following the kit protocol of PCR Tag, Bioline company (24).
Polymerase chain reaction set up:
Five taq red reaction buffer (10 μL).
Template (1 to 2 μL).
- Primers (20 μM each) (0.5 μL).
- –THO1 forward 5′-ATTCAAAGGGTATCTGGGCTCTGG-3′.
- –THO1 reverse 5′-GTGGGCTGAAAAGCTCCCGATTAT3′.
- –Amelogenin forward 5′-ACCTCATCCTGGGCACCCTGGTT3′.
- –Amelogenin reverse 5′-AGGCTTGAGGCCAACCATCAG3′. The size used 150 bp.
MyTaq HS Red Mix, 2x (12.5 μL).
Water (dH2O) (up to 25 μL).
The gene tyrosine hydroxylase 1 (TH01) has been suggested as a candidate for human longevity, also it is widely used in forensic genetics (25). TH01 also has many forensic medicolegal importance like its association with the occurrence of SIDS (Sudden death infant syndrome). It regulates gene expression and catecholamine production with allele 9.3 exerting a particularly strong effect on noreadrenaline production (26).
Amelogenin one of the important gene that determine the gender. The gender from any sample is always determined by the DNA profile based on the presence of Y allele in amelogenin in autosomal STR in single marker of Y chromosome. So, the detection of any single Y chromosome in an unknown sample proves that it belongs to a male individual (27; and 28).
Polymerase Chain Reaction Cycling Conditions
Polymerase chain reaction was carried out on the extracted DNA samples. The PCR was conducted as following; denaturation at 95 °C for 11 min; 28 cycles of denaturing at 94 °C for 1 min. Then annealing at 59 °C for 1 min. Extension at 72 °C for 1 min and finally extension at 60 °C for 60 min (29).
Analysis of amplification products
Amplification products of blood and semen DNA were compared to control using gel electrophoresis (29).
Ethical Approval
All ethical statements of The Research Ethics Committee, Faculty of Medicine, Assiut University, were followed. All participants were filled an informed consent after full explanation of the study and its aim. No risk for volunteers if they refuse to share in the study. The ethical approval number is 17200332.
Statistical Analysis
Data collected were analyzed using statistical program of social science version 20. The data were expressed as mean ± standard error for nonparametric values and standard deviation (SD) for parametric values. The difference between means was done using Student t test.
Results
All samples either blood or semen were analyzed to detect the effect of different grades of temperature and burn, firstly on the macroscopic appearance of the stains followed by presumptive tests to detect the nature of the specimen (blood or semen). Finally quantitative and qualitative analysis of DNA.
Effect of Different Grades of Temperature and Burn on Blood Stains
Macroscopic appearance of bloodstains after exposure to different grades of temperature and burn
Figure 1 shows the effect of exposure to different temperature grades (including burn) on the macroscopic appearance of the blood stain on the cotton fabric in comparison to the control. Blood stains exposed to temperature grades 100 °C and 50 °C became darker in color than the control. Burnt blood stains became charred. Blood stains exposed to temperature grades 37°C, 4°C, and −20°C did not show any change in their macroscopic appearance compared to the control.
Figure 1:
Effect of exposure to different grades of temperature and burn on macroscopic appearance of the blood stain on the cotton fabric in comparison to the control.
Presumptive test of bloodstains (Kastle-Mayer test) after exposure to different grades of temperature and burn
Figure 2 shows the effect of exposure to different temperature grades (including burn) on detection of the bloodstain on the cotton fabric by phenolphthalein test in comparison to the control. Bloodstains exposed to temperature grades 100 °C, 50 °C, 37 °C, 4 °C, and −20 °C gave positive results as strong in color as the control within 2 seconds of the test. Burnt bloodstains gave negative results and no color change was observed within 2 seconds of the test.
Figure 2:
Effect of exposure to different grades of temperature and burn on detection of bloodstain on the cotton fabric by phenolphthalein test (Kastle-Mayer test) in comparison to the control.
DNA concentration in blood stains after exposure to different grades of temperature and burn
Table 1 represents the effect of exposure to different temperature grades (including burn) on blood DNA concentration (ng/μL) measured by spectrophotometer in comparison to the control. There is highly significant reduction in the quantity of blood DNA extracted from bloodstains exposed to temperature grades 100 °C, 50 °C, and burn with mean value ± SD (52.28 ± 13.82, 65.64 ± 15.07, and 13.60 ± 2.77, respectively) compared to control. There was no significant reduction in the blood DNA concentration extracted from blood stains exposed to temperature 37 °C, 4 °C, and −20 °C with mean value ± SD (77.62 ± 8.01, 79.82 ± 3.58, 79.54 ± 3.82, respectively) compared to the control.
Table 1:
Effect of Exposure to Different Grades of Temperature and Burn on Blood DNA Concentration (ng/μL) Measured by Spectrophotometer in Comparison to the Control.
| Blood DNA conc (ng/µL) | p value | ||
|---|---|---|---|
| Mean | ±SD | ||
| Positive control | 80.92 | ±3.56 | |
| Temp 100 °C | 52.28 | ±13.82 | <0.001a |
| Temp 50 °C | 65.64 | ±15.07 | <0.001a |
| Temp 37 °C | 77.62 | ±8.01 | 0.10 |
| Temp 4 °C | 79.82 | ±3.58 | 0.33 |
| Temp −20 °C | 79.54 | ±3.82 | 0.24 |
| Burn | 13.60 | ±2.77 | <0.001a |
Abbreviation: SD, standard deviation.
a p value ≤ 0.001 highly significant.
Analysis of DNA primers by gel electrophoresis of blood stains after exposure to different grades of temperature and burn
Figure 3 represents PCR amplified product of THO1 primer and Amelogenin primer for positive and negative control of bloodstains by using 2% agarose gel electrophoresis stained with Ethidium Bromide (EthB). (Lane 1) 50 bp DNA marker. (Lane 2) Positive control by THO1 primer. (Lane3) Positive control by Amelogenin primer. (Lane 4) Negative control by THO1. (Lane5) Negative control by amylogenin. The bands in the lane 2 are two bands at 200 and 210 bp. The bands in lane 3 are three bands at 210, 230, and 60 bp. No bands appear in lane 4, 5.
Figure 3.
Two percent agarose gel electrophoresis stained with EthB showing PCR amplified product of THO1 primer and Amelogenin primer for positive and negative control of bloodstains. (Lane 1) 50 bp DNA marker. (Lane 2) Positive control by THO1 primer. (Lane 3) Positive control by amylogenin primer. (Lane 4) Negative control by THO1. (Lane 5) negative control by Amylogenin.
Figure 4 represents PCR amplified product ofTHO1 primer for the effect of exposure to different temperature grades (including burn) on detection of bloodstain on the cotton fabric in comparison to the control by using 2% agarose gel electrophoresis stained with EthB. (Lane 1) 50 bp DNA marker. (Lane 2) Positive control. (Lane 3) Bloodstain exposed to temperature grade 100 °C. (Lane 4) Bloodstain exposed to temperature 50 °C. (Lane 5) Bloodstain exposed to temperature 37 °C. (Lane 6) Bloodstain exposed to temperature 4 °C. (Lane 7) Bloodstain exposed to temperature −20 °C. (Lane 8) Burnt bloodstain. The bands in the lane 3, 4, 5, 6, and 7 appear as the control in the form of two bands at 200 and 210 bp. No bands appear in lane 8.
Figure 4:
Two percent agarose gel electrophoresis stained with EthB showing PCR amplified product of THO1 primer for the effect of exposure to different temperature grades (including the burn) on detection of blood stain on the cotton fabric in comparison to the control. (Lane 1) 50 bp DNA marker. (Lane 2) Positive control. (Lane3) Bloodstain exposed to temperature grade 100 °C. (Lane 4) Bloodstain exposed to temperature 50 °C. (Lane 5) Bloodstain exposed to temperature 37 °C. (Lane 6) Bloodstain exposed to temperature 4 °C. (Lane 7) Bloodstain exposed to temperature −20˚. (Lane 8) Bloodstain was burnt.
Figure 5 represents PCR amplified product of Amelogenin primer for the effect of exposure to different temperature grades (including the burn) on detection of blood stain on the cotton fabric in comparison to the control by using 2% agarose gel electrophoresis stained with EthB. (Lane 1) 50 bp DNA marker. (Lane 2) Positive control. (Lane 3) Bloodstain exposed to temperature grade 100 °C. (Lane 4) Bloodstain exposed to temperature 50 °C. (Lane 5) Bloodstain exposed to temperature 37 °C. (Lane 6) Bloodstain exposed to temperature 4 °C. (Lane 7) Bloodstain exposed to temperature −20 °C. (Lane 8) Burnt bloodstain. The bands in the lane 3, 4, 5, 6, and 7 appear as the control in the form of three bands at 210, 230, and 60 bp. No band appear in lane 8.
Figure 5:
Two percent agarose gel electrophoresis stained with EthB showing PCR amplified product of Amelogenin primer for the effect of exposure to different temperature grades (including the burn) on detection of blood stain on the cotton fabric in comparison to the control. (Lane 1) 50 bp DNA marker. (Lane 2) Positive control. (Lane 3) Bloodstain exposed to temperature grade 100 °C. (Lane 4) Bloodstain exposed to temperature 50 °C. (Lane 5) Bloodstain exposed to temperature 37 °C. (Lane 6) Bloodstain exposed to temperature 4 °C. (Lane 7) Bloodstain exposed to temperature −20 °C. (Lane 8) Bloodstain was burnt.
Effect of Temperature and Burn on Seminal Stains
Macroscopic appearance of seminal stains after exposure to different grades of temperature and burn
Figure 6 represents the effect of exposure to different temperature grades (including burn) on the macroscopic appearance of the seminal stain on the cotton fabric in comparison to the control. Semen stains exposed to temperature grades 100 °C, 50 °C, 37 °C, 4 °C, and −20 °C appear grayish white in color, the same as the control. Burnt semen stains became charred.
Figure 6:
Effect of exposure to different grades of temperature and burn on macroscopic appearance of the seminal stain on the cotton fabric in comparison to the control.
Presumptive tests of seminal stains (Florence test) after exposure to different grades of temperature and burn
Figure 7 represent the effect of exposure to different grades of temperature and burn on detection of seminal stain on the cotton fabric by Florence test. Seminal stains exposed to temperature grades 37 °C, 4 °C, and −20 °C gave positive results the same as the control in the form of brown rhombic shaped crystals seen by the light microscope. Seminal stains exposed to temperature grades 100 °C, 50 °C, and the burnt one gave negative results in the form of absence of brown shaped crystals by the light microscope.
Figure 7:
Effect of exposure to different grades of temperature and burn on detection of semen on the cotton fabric by Florence test in comparison to the control.
DNA concentration in semen stains after exposure to different grades of temperature and burn
Table 2 represents the effect of exposure to different grades of temperature and burn on semen DNA concentration (ng/μL) measured by spectrophotometer in comparison to the control. There was highly significant reduction in the concentration of DNA extracted from seminal stains exposed to temperature grades 100 °C and 50 °C with mean value ± SD (59.85 ± 13.28 and 65.06 ± 13.75, respectively) compared to the control. There was highly significant reduction in the concentration of semen DNA extracted from burnt seminal stains with mean value ± SD (13.65 ± 3.95) compared to control. There is no significant reduction in the semen DNA concentration extracted from seminal stains exposed to temperature 37 °C, 4 °C, and −20 °C with mean value ± SD (87.05 ± 9.87, 89.55 ± 4.11, 89.30 ± 4.23, respectively) compared to control.
Table 2:
Effect of Exposure to Different Grades of Temperature and Burn on Semen DNA Concentration (ng/μL) Measured by Spectrophotometer in Comparison to the Control.
| Semen DNA conc (ng/µL) | |||
|---|---|---|---|
| Mean | ±SD | p value | |
| Positive control | 90.90 | ±2.845 | |
| Temp. 100 °C | 59.85 | ±13.82 | <0.001a |
| Temp. 50 °C | 65.06 | ±13.75 | <0.001a |
| Temp. 37 °C | 87.05 | ±9.87 | 0.10 |
| Temp. 4 °C | 89.55 | ±4.11 | 0.23 |
| Temp. −20 °C | 89.30 | ±4.23 | 0.16 |
| Burn | 13.65 | ±3.95 | <0.001a |
Abbreviation: SD, standard deviation.
a p value ≤ 0.001 highly significant.
Analysis of DNA primers by gel electrophoresis of semen stains after exposure to different grades of temperature and burn
Figure 8 represents PCR amplified product of THO1 primer and Amelogenin primer for positive and negative control of seminal stains. (Lane 1) 50 bp DNA marker by using 2% agarose gel electrophoresis stained with EthB. (Lane 2) Positive control by THO1 primer. (Lane 3) Positive control by Amylogenin primer. (Lane 4) Negative control by THO1. (Lane 5) Negative control by Amylogenin. The bands in lane 2 are two bands at 200 and 210 bp. The bands in lane 3 are three bands at 210, 230, and 60 bp. No bands appear in lanes 4 and 5.
Figure 8:
Two percent agarose gel electrophoresis stained with EthB showing PCR amplified product of THO1 primer and Amelogenin primer for positive and negative control of semen stains. (Lane 1) 50 bp DNA marker. (Lane 2) Positive control by THO1 primer. (Lane3) Positive control by Amelogenin primer. (Lane 4) Negative control by THO1. (Lane5) Negative control by Amylogenin.
Figure 9 represents PCR amplified product of THO1 primer for the effect of exposure to different grades of temperature and burn on detection of seminal stain on the cotton fabric in comparison to the control by using 2% agarose gel electrophoresis stained with EthB. (Lane 1) 50 bp DNA marker. (Lane 2) Positive control. (Lane 3) Seminal stain exposed to temperature grade 100 °C. (Lane 4) Seminal stain exposed to temperature 50 °C. (Lane 5) Seminal stain exposed to temperature 37 °C. (Lane 6) Seminal stain exposed to temperature 4 °C. (Lane 7) Seminal stain exposed to temperature −20 °C. (Lane 8) Seminal stain was burnt. The bands in lanes 3, 4, 5, 6, and 7 appear as the control in the form of two bands at 200 and 210 bp. No bands appear in lane 8.
Figure 9:
Two percent agarose gel electrophoresis stained with EthB showing PCR amplified product of THO1 primer for the effect of exposure to different grades of temperature and burn on detection of seminal stain on the cotton fabric in comparison to the control. (Lane 1) 50 bp DNA marker. (Lane 2) Positive control. (Lane 3) Seminal stain exposed to temperature grade 100 °C. (Lane 4) Seminal stain exposed to temperature 50 °C. (Lane 5) Seminal stain exposed to temperature 37 °C. (Lane 6) Seminal stain exposed to temperature 4 °C. (Lane 7) Seminal stain exposed to temperature −20 °C. (Lane 8) Seminal stain was burnt.
Figure 10 represents PCR amplified product of Amelogenin primer for the effect of exposure to different grades of temperature and burn on detection of seminal stain on the cotton fabric in comparison to the control by using 2% agarose gel electrophoresis stained with EthB. (Lane 1) 50 bp DNA marker. (Lane 2) Positive control. (Lane3) Blood stain exposed to temperature grade 100 °C. (Lane 4) Seminal stain exposed to temperature 50 °C. (Lane 5) Seminal stain exposed to temperature 37 °C. (Lane 6) Seminal stain exposed to temperature 4°C. (Lane 7) Seminal stain exposed to temperature −20°C. (Lane 8) Seminal stain was burnt. The bands in lanes 3, 4, 5, 6 and 7 appear as the control in the form of three bands at 210, 230, and 60 bp. No bands appear in lane 8.
Figure 10:
Two percent agarose gel electrophoresis stained with EthB showing PCR amplified product of Amelogenin primer for the effect of exposure to different grades of temperature and burn on detection of semen stain on the cotton fabric in comparison to the control. (Lane 1) 50 bp DNA marker. (Lane 2) Positive control. (Lane 3) Seminal stain exposed to temperature grade 100 °C. (Lane 4) Seminal stain exposed to temperature 50 °C. (Lane 5) Seminal stain exposed to temperature 37 °C. (Lane 6) Seminal stain exposed to temperature 4 °C. (Lane 7) Seminal stain exposed to temperature −20 °C. (Lane 8) seminal stain was burnt.
Comparison Between the Stability of Bloodstain Samples and Seminal Stain Samples After Exposure to Different Grades of Temperature and Burn:
Presumptive tests of blood and semen stains after exposure to different grades of temperature and burn
Table 3 represents comparison between the effect of different grades of temperatures and burn on detection of blood and seminal stains by presumptive tests (phenolphthalein for blood stain and Florence test for semen stain). Temperature grades 100 °C and 50 °C gave negative results for seminal stains but not for blood stains compared to control. Temperature grades 37 °C, 4 °C, and −20 °C did not affect the results (gave positive results) of both blood and seminal stains compared to control. Burn affect the results (gave negative results) of both blood and seminal stains compared to control.
Table 3:
Comparison Between the Effect of Different Grades of Temperature and Burn on Detection of Blood and Seminal Stains by Presumptive Tests (Phenolphthalein for Bloodstain and Florence test for Seminal Stain).
| Control | Tem 100 °C | Temp 50 °C | Temp 37 °C | Temp 4 °C | Temp −20 °C | Burn | |
|---|---|---|---|---|---|---|---|
| Phenolphthalein test | +ve | +ve | +ve | +ve | +ve | +ve | −ve |
| Florence test | +ve | −ve | −ve | +ve | +ve | +ve | −ve |
DNA concentration
Figure 11 represents comparison between level of DNA concentration percentage in blood and seminal stains after exposure to different grades of temperatures and burn. There was significant difference at P value ≤ 0.01 in the percentage of concentration of DNA between blood and seminal stains when exposed to temperature grade 50 °C, with higher DNA concentration obtained from blood samples. There was no significant difference in the concentration of DNA between blood and seminal stains when exposed to temperature grades 100 °C, 37 °C, 4 °C, −20 °C, and burn.
Figure 11:
Comparison between mean concentration level of DNA in blood and seminal stains after exposure to different grades of temperatures and burn.
Analysis of DNA primers by gel electrophoresis
Table 4 represents comparison between blood and semen after exposure to different grades of temperature and burn by THO1 and Amelogenin primer analysis. Both blood and seminal stains can be detected by using THO1 and Amelogenin primer except those exposed to burn.
Table 4:
Comparison Between Blood and Seminal Stains After Exposure to Different Grades of Temperature and Burn by THO1 and Amelogenin Analysis.
| Temp. 100 | Temp. 50 | Temp. 37 | Temp. 4 | Temp. −20 | Burn | ||
|---|---|---|---|---|---|---|---|
| THO1 | Blood | +ve | +ve | +ve | +ve | +ve | −ve |
| Semen | +ve | +ve | +ve | +ve | +ve | −ve | |
| Amelogenin | Blood | +ve | +ve | +ve | +ve | +ve | −ve |
| Semen | +ve | +ve | +ve | +ve | +ve | −ve |
Discussion
When fires or explosion set in the crime scene, the scene will be difficult to process. The investigators will face difficulty in recognizing, locating, and identifying the evidence among the artefacts of the fire or explosion (30).
The exposure to different grades of temperature and burn may affect the bloodstain at the level of macroscopic appearance, identification by preliminary tests, and DNA analysis. In the current research, the stains exposed to temperature grades 100 °C and 50 °C for 24 hours showed change in the color of blood stain to become darker than the control stain. This may be attributed to change the concentration of methaemoglobin, these results are in agreement with results of McDonald (31). Burnt blood stains became charred and black as reported by Klein et al (19).
The influence of the temperature on the ability to identify bloodstains by presumptive test (phenolphthalein) was investigated in the current study and the results showed that blood could be detected from the bloodstain on cotton fabric by phenolphthalein test after exposure to temperature grades 100 °C, 50 °C, 37 °C, 4 °C, and −20 °C. As the positive results of the phenolphthalein test depend on the presence of oxidase enzyme which is not affected by different grades of temperature. These findings are in close agreement with Khushbu et al. (8) who investigated the ability to identify bloodstains under different environmental conditions (temperature grades 28 °C, 4 °C, −20 °C, 150 °C, and different acidic and alkaline pH condition). Burnt blood stains could not be identified by phenolphthalein test in the current study due to complete charring of the stain. This is in accordance with the results of Vineyard et al. (32). This could be explained as high temperature causes oxidation of Fe+ 2 to Fe+ 3. Therefore, no color change would be observed in the absence of Fe+ 2 (8).
In the present study, the quantity of extracted DNA from bloodstains exposed to temperature grades 100 °C, 50 °C, and burn was significantly reduced. This is attributed to the degradation of the proteins which lead to degradation of DNA (33). Another opinion for Karni et al., (34) who reported that in their studies, degradation of DNA begins in dry conditions at 130 °C and complete degradation occurs in 190 °C, while in water degradation begins above 90 °C, it is possible that the pressure weakness the chemical bonds between the atoms within the DNA molecules. These results coincided with observations of Barbaro and Cormaci (5) who stated in their study about the effect of high temperatures on STRs typing that high temperature of the biological samples causes decrease in DNA concentration. Opposite to the present results, other studies performed by Klein et al. (19) mentioned that DNA has a very stable structure and can be detected after exposure to high temperatures even if the previous testing for blood has been negative. This is may be due to using other substrates rather than cloth as they used metals also, due to using different methods of extraction as they used automated extractor (Maxwell® 16 System, Promega), for DNA extraction and DNA profiling by (PowerPlex® 16 ESI/ESX, Promega).
The results obtained in the study of Al-Kandari et al. (4) showed a significant association between the temperature and the quantity (ng/µL) of DNA. High temperature can denature DNA, causing the double helix to split resulting in 2 single stranded DNA molecules which lead to degradation of DNA.
Low temperature has the power to preserve the quantity of DNA in biological samples (35). In the current study, maximum DNA concentration could be recovered from bloodstains exposed to temperature grades 37 °C, 4 °C, and −20 °C, without significant difference from the positive control. These findings are in concordance with previous studies such as Aiflokayan (18) and Ng et al. (17) who studied the effects upon DNA concentration after storage of samples in different temperatures. They extracted the sample using DNA IQTM Casework Extraction Kit on a Promega Maxwell1 16 instrument and measured the concentration using the Quantifiler1 Duo DNA Quantification kit on the Applied Biosystems 7500 Real-Time PCR system.
The current study revealed that DNA quality did not significantly affected by temperature grades 100 °C, 50 °C, 37 °C, 4 °C, and −20 °C. DNA fragments could be detected by using gel electrophoresis of PCR amplified products of THO1 primer and Amelogenin primer. This finding could be explained by the capability of STR markers to generate typing results from very degraded material. This finding is in agreement with previous studies such as Dissing et al. (35) in their study about the stability of DNA in stains at extreme temperature. They found that blood DNA can be detected even after degradation by the effect of high and low temperature as the amount of DNA was enough for performing PCR. As regard the burnt blood samples in the present study DNA cannot be detected by using gel electrophoresis of PCR amplified products of THO1 and Amelogenin primers due to the charring of the sample and formation of soot which can interfere with the ability to obtain DNA profile. This was in agreement with Tontarski et al. (6) in their study about DNA recovery after fire exposure. They found that no DNA profiles were obtained from the burnt samples.
In the current study, seminal stains exposed to temperature grades 100 °C, 50 °C, 37°C, 4°C, and −20 °C for 24 hours appear grayish white in color the same as the control stain. Burnt seminal stains became charred.
Presumptive test (Florence test) was performed to identify seminal stains after exposure to different grades of temperature and burn. The current study showed that choline per iodide crystals (brown rhombic shaped crystals) could be detected by Florence test after exposure to temperature grades 37 °C, 4 °C, and −20 °C and cannot be detected by Florence test for samples exposed to 100 °C, 50 °C, and the burnt samples due to complete charring of the samples that interfere with the ability to form choline per iodide crystals.
The amount of recovered DNA from seminal stains exposed to temperature grades 100 °C, 50 °C, and burn was significantly reduced. The explanation of this result is due to degradation of DNA which is parallel to degradation of proteins by high temperature. These results also reported by Al-Kandari et al. (4) and Snyder and Aldredge (36) in their study about temperature effects on DNA quantity, they stated that high temperatures can change significantly the state of DNA stability thus reducing its survival. Maximum DNA concentration could be recovered from seminal stains exposed to temperature grades 37 °C, 4 °C, and −20 °C, without significant difference from the positive control as low temperature is ideal for preservation of DNA. This finding is in concordance with previous studies as Raina et al. (16) who reported in their study about the effect of storage conditions of seminal stains on cloth that DNA concentration did not affected by low temperatures.
Regarding the quality of isolated DNA used in PCR amplification reaction, gel electrophoresis of seminal stains shown in this study, revealed that DNA quality did not significantly affected by temperature grades 100 °C, 50 °C, 37 °C, 4 °C, and −20 °C, and DNA fragments could be detected by using gel electrophoresis of PCR amplified products of THO1 primer and Amelogenin primer. This finding could be explained by the capability of STR markers generate typing results from very degraded material. This finding is in concordance with previous studies as (4) in their study about the effect of temperature on semen DNA. They found that semen DNA can be detected even after degradation by the effect of high and low temperature as the amount of DNA was enough for performing PCR. DNA cannot be detected by using gel electrophoresis of PCR amplified products of THO1 and Amelogenin primers for burnt seminal stains in the present results. This is attributed to complete charring of the stain and accumulation of soot that would interfere with successful extraction of DNA, this finding is also explained by Tontarski et al. (6) in their study about DNA recovery from blood stains after fire exposure.
Comparing between the stability of the blood and seminal stains, the present study showed that blood can be identified in bloodstains exposed to temperature grades 100 °C and 50 °C by presumptive test (phenolphthalein), while semen cannot be identified by presumptive test (Florence) in seminal stains exposed to the previous grades of temperature. The exposure of blood and seminal stains to temperature grades (37 °C, 4 °C, −20 °C and burn) resulted in no significant difference between blood and seminal stains as regard their detection by presumptive tests.
The quantity of extracted DNA from bloodstains was compared with that from seminal stains in the present study. There were no significant differences between the percentage of DNA which extracted from bloodstains and seminal stains exposed to all temperature grades (100 °C, 37 °C, 4 °C, −20 °C) except temperature grade 50 °C as higher percentage of DNA in bloodstain was detected compared to the percentage of DNA in seminal stains. This agreed with Barbaro and Cormaci (5) in their study about the effect of high temperature on STRs typing, they reported that blood samples gave better results than seminal samples. These findings are opposite to the results of Al-Kandari et al. (4) in their study about the effect of temperature on the quantity of DNA in blood and semen. They stated that DNA in semen samples were resistant to degradation at temperature grade than blood.
In the current study, there was no significant difference between DNA concentration extracted from blood and seminal stains exposed to high temperatures (100 °C and burn) and degradation occurred in both types of samples. Snyder and Aldredge (36) proved that in their study, DNA degrade in both blood and semen stains after fire exposure.
Comparing between the two primers used in the present study (TH01 and Amelogenin), the results showed that bot primers could be used successfully to detect DNA after exposure to different degree of temperature.
Conclusion
In conclusion, different grades of temperature and burn are considered as risk factors for stains analysis. It can lead to misinterpretation and may lead to escape of the assailant from the law.
In the current study, the authors tried to prove this hypothesis to find the best test that help in identification of the stains in the crime scene. Macroscopic changes of the stains either blood or semen and presumptive tests for both stains can give idea about the different grades of temperature and burn that the stain exposed to, but not confirmatory.
DNA analysis especially quantification of the concentration and gel electrophoresis help to determine that the stain exposed to different grades of temperature and burn, and degradation occurred. Both, macroscopic changes and DNA analysis can help analysist to know the effect of different grades of temperature and burn on the stain in the crime scene.
Acknowledgments
Molecular and genetic lab in the university—DNA forensic and diagnostic lab in the faculty of Medicine.
AUTHORS
Randa H. Abdel Hady, Professor of Forensic Medicine and Clinical Toxicology Department, Assiut University
Roles: Share in the selection of the research point, share in writing of the research article, revise the article before publication.
Hayam Z. Thabet, Professor of Forensic Medicine and Clinical Toxicology Department, Assiut University
Roles: Share in the statistics part of the study, share in writing of the research article, revise before publication.
Noha Esmael Ebrahem, Professor of Forensic medicine and clinical Toxicology Department, Assiut University
Roles: Collection of the samples, share in the statistics of the research, and writing of the paper.
Heba A. Yassa, Lecturer of Forensic Medicine and Clinical Toxicology Department, Assiut University
Roles: Share in the selection of the point of research, in the lab work, writing the research article.
Footnotes
Availability of Data and Materials: All data is available on request.
Consent for Publication: All authors approve the publication and revise the research before sent to the journal.
Ethics Approval and Consent to Participate: All ethical considerations about using samples and informed consent from participants were taken, with approval from the ethical committee—Assiut University faculty of medicine.
Statement of Human and Animal Rights: All ethical consecrations in dealing with human volunteers were followed during the research.
Disclosures & Declaration of Conflicts of Interest: The authors, reviewers, editors, and publication staff do not report any relevant conflicts of interest.
Financial Disclosure: Fund of this research is from the funding unit in the faculty of Medicine—Assiut University.
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