Human DNA Profiling: To Evaluate Different Techniques For A DNA-Sample To Obtain DNA-Profile For A Mock-Sex-Assault
DNA analysis is highly important in the field of forensic science. Forensic scientists/ researchers/ investigators can compare DNA profiles of biological evidence samples with a data bank of DNA or to the control sample to assist the police in detecting suspects. The amount of DNA, quality of a sample, its handling, storage, and processing will make difference to the outcome obtained from that sample DNA- material. DNA-analysis consists of many step by step sub-procedures as the sampling of DNA material, extraction of DNA, quantization of DNA, amplification of DNA and profiling of DNA. There is a wide range of instruments and commercial kits are available in the market serving a variety of purposes as per the requirements. We performed a mock sex-assault to obtain a DNA profile via undergone different techniques for sampling, extraction, quantization and amplification purposes. We conducted tape lift for the sampling process which was followed by the extraction with the Chelex Resin and Qiagen DNeasy Blood & Tissue Kit. The Quantifiler Trio qPCR setup and Qubit 3 Fluorimeter Quantification were observed for the quantization process of the obtained sample from the extraction method and the Quantifiler Trio qPCR noticed to be more effective in our results. Amplification and capillary electropherogram were performed with PowerPlex 21 System and 3130xL systems respectively. After completing the processing, the DNA profile was analyzed with the help of GeneMarker software and the DNA sample was found to be a mixture of male and female DNA. The whole process should be performed with the least chances of contamination and proper training of the handling instruments and other variety of equipment. The experimenter should adhere to the laboratory protocols and the user’s manual of the products to obtain the best results.
Introduction
In today's date, Forensic Researchers are being able to analyze too little biological sample to prepare DNA-profile of the sample due to the advancement of technology. A very low-level DNA sample- which is known as “Touch-DNA or Trace-DNA” - from which recovery of fingerprint might not be effective; examination of DNA would help a lot. Sex assaults are one of the major types of crime where Trace-DNA evidence plays a pivotal role to assist in the investigation. According to the guidelines for Evidence-based Forensic based sampling from the RPCA (The Royal College of Pathologists of Australia), has provided different samplings as per the circumstances and severity of the assault. For the sampling process, tape-lifting is a very well-established, convenient and efficient method for biological/DNA materials, fabric traces, solid surface and/or skin. Sampling with Tapelift can lead to a mixture of DNA-profile. DNA profile of mixture refers to a DNA profile that is originated from two or more donors for the sampled biological material. After sampling with tape-lift, extraction of DNA sample was performed with the use of two different methods- Chelex-Resin and Qiagen DNeasy Blood and Tissue kit. A Chelex-based protocol is a fast, practical, and effective method for extracting DNA of high quality and with low contamination, which does not involve organic solvents. The Qiagen DNeasy Blood and Tissue kit are well-established for the purification of total DNA from animal blood, animal tissue, bacterias, and insects.
The Quantifiler Trio qPCR setup and Qubit 3 Fluorimeter Quantification were observed for the quantization process in which Qubit 3 Flourimeter is very compact and easy to use but it can not detect too low concentrations which is a contrast to Quantifiler Trio qPCR technique. “Polymerase Chain Reaction (PCR) is a simple, yet elegant, enzymatic assay, which allows for the amplification of a specific DNA Fragment from a complex pool of DNA”. Despite its wide applicability, the success rate of the PCR method depends on the quality and quantity of the DNA template, which should be free of contaminants and DNA nucleases that impair the amplification process. Capillary Electrophoresis(CE) can be used forensically to separate the blood contents such as DNA, RNA, inorganic substances according to their mass.
To analyze the DNA-profile obtained from the sample, technology is moving fast and making them easier than ever before. GeneMarker software is one of the best software for analysis of a DNA profile due to its swiftness, integration towards the latest technology, and ease of operation. Besides the uniqueness of DNA and easiness of obtaining DNA profile from a very little DNA sample, there are so many conditions and inhibitors such as contamination of the sample, interference of some chemicals, degradation of effect on DNA that can harass the above-mentioned task.
Methods and Materials
Collection of the sample by Tape lifting Method: To generate the simulated assault victim sample containing both male and female DNA, a volunteer male and volunteer female donated their DNA sample simulated as “offender” and “victim” respectively. The Victim’s wrist was held firmly by the Offender and two other volunteers tried to move the holding arm for about 20 seconds. The victim was then allowed to sit without disturbing the grappled area for 5 mins and then the tape-lifting of the wrist area of the victim was processed and stored into a petri dish. The area of tape containing cellular material was divided into two even halves and each half was placed each half into an Eppendorf tube and were labeled as Sample 5 and Sample 6.
DNA Extraction Process: We Added 500 μl of 5% Chelex® solution which was well-mixed just before adding; to the DNA containing sample. Then added 10ul Proteinase K with 20mg/ml concentration. We incubated at different temperatures after giving vortex each time as per the laboratory guidelines. Then we transferred the supernatant (containing DNA) into a clean, 1. 5ml tube leaving all the Chelex® beads or other solid material using a pipette.
DNA Quantization: 12 PCR Tubes were prepared as per the guidelines. the following Quantifiler Trio Standards in PCR tubes using the provided 50 ng/uL standard was prepared and the Quantifiler THP DNA Dilution buffer was added. The 12 PCR reaction tubes pipette were treated with 5uL of the Quantifiler Trio reaction mix and 4uL of the Quantifiler Trio primer mixture of 1uL of calibrators, samples and reagent blank (ultrapure water) to each reaction tube was introduced in such a way that no bubbles were there. After placing tubes on ice. The further analysis of this tubes was undertaken with QuantStudio6 flex qPCR Instrument. The 10 folds of dilutions were performed o the standard samples. To draw the standard curve graph for CT values and Concentrations; the natural logarithm of the concentrations was taken into account and the standard curve was drawn for the CT against log Concentrations.
To prepare Qubit working solution, we took 1194uL of Qubit dsDNA HS Buffer and 6uL of Qubit dsDNA HS Reagent in labeled 1. 5 mL tube. We pipette out 190 uL of the prepared Qubit working solution and added 10uL of your sample DNA extracts to that. Mixed them well and took readings with the instrument.
Polymerase Chain Reaction (PCR): The PCR reaction was performed with the PowerPlex 21 System. All the procedures were performed as per the guidelines of the user manual of the PowerPlex 21 System.
Capillary Electrophoresis: This process was undertaken with the help of the 3130xl instrument and all actions were performed under the guidance and directions of the User-manual.
Data Analysis of the DNA Profile: GeneMarker software was used to analyze the obtained DNA profile. Their user-manual directed for the necessary settings and comparison purposes of the obtained results/peaks.
Discussion
The results collected allow us to compare the two major extraction techniques; the Chelex® Resin Method and the Qiagen DNeasy Blood & Tissue Kit method. Extraction of a DNA sample generally consists of the three basic steps which are 1) Lyse (break open) the cells; 2) Separate the DNA from the other cell components, and 3) Isolate the DNA. “Chelex1-100 (Bio-Rad Laboratories, CA, USA) is a chelating resin which uses ion exchange to bind transition metal ions” which is made up of Styrene Divinylbenzene copolymers along with paired iminodiacetate ions which provide the binding site for the polyvalent metal ions. “During the extraction process, the alkalinity of the solution and the act of boiling the solution breaks down the cells and allows the chelating groups to bind to the cellular components, protecting the DNA from degradation”.
The Chelex Resin Method advocated as it is quick, it does not require multiple tube transfers and it does not use toxic organic solvents such as Phenol-Chloroform but on the contrary, it is not efficient to eliminate inhibitors (such as haem) which can be detrimental to downstream processes. According to the Life Science News Article “the Qiagen DNeasy Blood & Tissue Kit allows for fast and easy purification of total DNA from a variety of sample sources, including fresh or frozen animal and plant tissue or cells, blood, bacteria, yeast, or viruses. In addition to that, the purified DNA is free from the majority of the inhibitors, proteins, nucleases, and other contaminants. “Therefore, the purified DNA can be used for a variety of downstream applications such as PCR, Southern hybridization, RAPD, AFLP, RFLP, sequencing, and cloning. DNeasy purified DNA is up to 50 kb in size, with fragments of approximately 30 kb size predominating; very small DNA fragments of 100 bp are also recovered”.
On the flip side of this, it is a very expensive kit and also it allows to copurify DNA and RNA when both are present in the sample. “The RNA does not affect PCR but may inhibit other downstream enzymatic reactions”. The standard curve which is obtained from the instrument QuantStudio6 flex qPCR is a graph of the CT values against the initial concentrations. The value of CT can be given as the following equation: CT = m [log (Qty)] + b; where m is the slope, b is the y-intercept, and Qty is the starting DNA quantity(12). As per the product manufacturers’ guide, R2 value ≥ 0. 99 indicates a close fit between the standard curve regression line and the individual CT data points of quantification standard reactions. In order to investigate the sensitivity of the Quant Trio kit, according to the user’s manual, a plot of the CT values versus the known DNA quantities showed the expected log-linear relationship between the two quantities - All dilutions, including samples at the lowest concentration (16 pg/μL), gave positive results for the Quantifiler Human kit-in which each dilution series, the data points form acceptable standard curve. The Qubit3 Fluorimeter dsDNA High Sensitivity instrument has 10 pg/L to 70 ng/L range for the concentration of starting sample.
Thus, the results which shows “out of range” and “sample concentration is TOO LOW” may be too high and too low concentrations respectively in compare to the given range from the manufacturers’. The obtained DNA-profile with the GeneMarker shows different peaks for the samples as shown in figures. One of the negative control can be interpreted as a result of the contamination which shows the presence of peaks; while another negative control has no contaminated peaks present.
The presence of X and Y peak in the same DNA profile can be interpreted as a “mixture DNA profile”. The control sample shows large contamination that may be a result of poor handling and storage. To improve the quality of results, contamination must be avoided and each and every step should be performed as per the guidelines of the product manufacturers. In most cases, human errors and instrumental errors play a pivotal role in the diversion of the results in profiling DNA samples.
To ensure the proper results there is a range of factors and measures you need to follow while pursuing the experiment. A very small inaccuracy may result in an unexpected error in results. Choice of correct method, proper training, contamination-free environment, and handling techniques, together, can lead to a successful DNA profiling, even with a too low amount of sample DNA with the advanced technologies.
Conclusion
DNA profiling of too little biological material is now available and that has made vast differences into the field of forensic science, but it comes with challenges too. The choice of technique for every step should be according to the sample range/concentration and as per the protocols of the manufacturer.
