Forensic Analysis of DNA and Biological Material Report

Aim

The first aim of this report was to undertake the isolation, quantification, amplification and analysis of Deoxyribonucleic acid (DNA) and biological materials, particularly buccal swabs. Secondly, the experiment aimed at familiarizing the person with the methodologies associated with DNA analysis.

Introduction

The Deoxyribonucleic acid (DNA) test was complex examination of biological examination, but necessary in legal cases to examine the person’s paternity. It could also help the forensic experts produce the evidence in cases of sexual assault since the DNA has distinct chemical properties to a particular person (Bieber et al., 2006, p. 1315). The unique features of the DNA properties of people make it useful and reliable in forensic investigations. Biomaterials such as semen, bone tissues, blood, and urine are widely used in carrying out forensic investigation on suspected criminals. This is made possible using the latest technological machines such as Nanodrop Spectrophotometer to enhance accuracy and reliability.

Method

In conducting the DNA test, the practical work follows four significant procedures. First, the forensic expert collected buccal swabs and extracted DNA using a solid phase technique called QIAGEN (Schafer 2008, p. 40). Second, the expert verified the DNA that was extracted and quantified it. Third, the expert carried out the PRC reaction on the collected DNA. Finally, the researcher analyzed the PRC products using gel electrophoresis (Saks & Faigman 2008, p. 150).

Practical Schedule 1: Isolation and Amplification of Genomic DNA

This was the first stage when carrying out the DNA test on a biological material. Here, the forensic analyst isolated DNA from a number of biological matrices, which for this experiment was buccal swab. Notably, this biological matrix was most commonly used in DNA tests. The process of taking a buccal swab and DNA extraction proceeded in a series of steps as follows.

• Step I: The Omni-swab packaging was opened at the handle end, and the swab carefully removed. Notably, the forensic analyst was not allowed to touch the collection pad of the swab as a precaution measure (Schafer 2008, p. 40).
• Step II: The researcher then held the handle end of the Omni-Swab and scraped the collection pad firmly against the inside of the cheek 6 times, being careful not to eject the tip. This was equally a precaution to avoid pouring the content or mixing them wrongly (Joseph 1989, p. 32).
• Step III: After taking the sample, the researcher ejected the pad into a labelled 1.5 ml micro-centrifuge tube by pressing the handle towards the collection pad.
• Step IV: In this step, 400μl phosphate buffered saline (PBS) was pipetted into the sample and centrifuged thoroughly.
• Step V: 14μl QIAGEN protease solution and 400μl of buffer AL was added to the sample tube and mixed by vortexing for 15 seconds. This was to make the substances mix properly.
• Step VI: The mixture was incubated at 56^oC for 10 minutes to give a good yield in the subsequent stages.
• Step VII: At this step, the sample was briefly centrifuged to remove droplets from the top of the lid. The aim of this process was to remove impurities, which could be in the mixture.
• Step VIII: 400μl ethanol was then added to the sample tube and mixed again for 15 seconds. Again, the sample tube was briefly centrifuged to remove droplets from the inside of the lid.
• Step IX: Here, 700μl of the mixture from step 8 was carefully pipetted into a QIAmp spin column (sitting in a 1.5 ml collection tube) without wetting the rim. The cap was then closed and centrifuged at 6000g for 1 minute (c 8000 rpm). After this, the flow was discarded through the collection tube for the next stage in the experiment.
• Step X: The QIAmp spin column was placed in a new 1.5ml collection tube. After this, the forensic analyst opened the spin column and added 500μl Buffer AW1 without wetting the rim. He closed the cap and centrifuged at 6000 g for 1 minute then discarded the flow through the collection tube.
• Step XI: The QIAmp spin column was placed in a new 1.5ml collection tube. Then, the spin column was opened and 500μl Buffer AW2 added to it without wetting the rim. The cap was closed and rotated at 20000g (13000 rpm) for 4 minutes. This procedure was to dry the QIAmp membrane. This was followed by discarding the flow through the collection tube.
• Step XII: The QIAmp spin column was placed in a clean 1.5ml micro-centrifuge tube. After which, the QIAmp spin column opened and 150μl Buffer AE added to it. The mixture was incubated at room temperature for 1 minute and then centrifuged at 6000g (c 8000 rpm) for 1 minute. This was done to elute the DNA into the micro-centrifuge tube. One precaution that the researcher had to take was not to throw away the micro-centrifuged tube because it contained the DNA sample.
• Step XIII: After this, the sample was labelled.

Practical Schedule 2: Verification and Qualification of DNA in the Extracted Sample

This was the second stage in analyzing the DNA and Biomaterials and proceeded in two parts with the following steps.

• Part 1: Yield gel and verification of the presence of high molecular weight DNA.
  • Step I: DNA sample was retrieved and fully defrosted, while the gels were already prepared with 1% agarose in TBE buffer. It was important to note that TBE buffer is a buffer prepared from Tris (hydroxymethyl) amino ethane (216g), Boric Acid (110g) and EDTA (18.6g). The buffer chemicals were then dissolved in 1800mL distilled water and the pH adjusted to pH 8.0 using HCl or NaOH as appropriate. This was then diluted tenfold (1 part buffer to 9 parts distilled water) before it could be used.
  • Step II: 2.5ul of DNA and 2.5ul of loading dye were pipetted into a small PCR tube. After this, a fresh pipette tip was used to withdraw and eject the liquid several times until it is well mixed. Here, the loading dye was prepared from buffer, glycerol and bromophenol blue, after which, a Lambda ladder was loaded on to the gel.
  • Step III: The gel with 5ul of DNA sample was carefully loaded using a pipette. The tip of the pipette was then placed into the agarose gel and slowly used in ejecting the solution taking care not to withdraw the tip.
  • Step IV: Once all of the samples were loaded onto the gel, electrophorese for about 1.5 hours using a gel voltage of 70 V, the forensic expert then expected to view the gel under UV light to visualise the DNA extracted from the sample (Saks & Faigman 2008, p. 157).
• Part 2: Determination of the Yield and Purity of the DNA Extracted from the Buccal Swab

Here, the expert made a 10x dilution of the template by adding 10uL of purified DNA to 90uL of HPLC grade water in a UV micro-cuvette. Then, he used HPLC water as buffer for a blank. At this level, the forensic analyst needed one blank per five samples (Bruce & Angela 2009, p. 340). Once this was through, the cuvettes were placed in the appropriate ports ensuring they face in the correct direction, for instance, with the clear part of the cuvette in line with the light beam (Bruce & Angela 2009, p. 341).

Practical Schedule 3: PCR Amplification of Extracted DNA

This was the third stage of the analysis and it proceeded in the following steps. Initially, the forensic analyst was given a master mix which consisted of the following:

2.5 ul of 10X PCR buffer (Tris-HCl, KCl, (NH₄)₂SO₄, 15 mM MgCl₂ (pH8.7)

1.5 ul 1.5 mM MgCl₂

1.0 ul 10mM primer CYTb1

1.0 ul 10mM primer CYTb2

0.2 ul of 25 mM dNTP’s (dATP, dTTP, dCTP and dGTP)

0.1 ul 5U/ul Taq DNA polymerase

17.7 ul sterile distilled water.

Notably, the reaction mixture and template DNA was done to give a final volume of 25 ul that would be used in the subsequent stages of the experiment. The researcher took 24ul of the master mix and put it in to a PCR tube. Then, 1ul of the prepared DNA was put it into the PCR tube with the master mix and centrifuged. This process was followed by programming the thermal cycler as follows, after which the mixture was held at 4^oC to get the best outcome, failure to which the researcher gets a false results (Nick 2002, p. 48).

Initial denaturation: 94^oC for 2 minutes

30 cycles: 94^oC for 30 seconds

55^oC for 30 seconds

72^oC for 1 minute

Final extension: 72^oC for 5 minutes

Practical Schedule 4: Analyse the PCR Products using Gel Electrophoresis

PCR GEL-1 (GROUP-1)

This was the final stage of forensic investigation and proceeded in the following steps. First, the PCR product was retrieved; then, 4ul of loading dye was pipetted directly into your PCR product tube. With draw and eject the liquid several times until it was well mixed. After this, the gel was loaded using the entire PCR product. Alongside the samples, a sample of Hyper Ladder 1 was run to allow the forensic analyst to determine the size of the amplified DNA (Gosline 2005, p. 12).

Results

PRACTICAL 23^rd FEB.

From the practical experiment, especially part 2, the results were as:

DNA ratio = 1.497

DNA = 104.1 Ng/mL

Proteins = 748.5 Ng/mL

Plank was water

Wavelength = 260 – 280

Absorbance = 0.267 – 0.178

Sample 1 was my sample = 0.26/0.17=1,529

DNA = 260/280 =1.8-2

Discussion

Based on the examination of the outcome of the experiment, it was apparent that as the distance covered increases, the DNA size increases, showing the reverse response of the DNA elements. In addition, the DNA ratio of 1.49 indicated that as the distance travelled increases, the size of the DNA molecule increases because of the disperse convexity. The contributing factors to this kind of movement were from the visual difficulties of viewing the DNA as a result of the staining method used. The discrete bands were observed when there was enough DNA materials present bind the dye to make it visible, which via the UV light makes stretched the DNA material to increase in size. Additionally, the proteins results are 748.5 Ng/ml when compared to the DNA ratio imply that the protein level increase in the DNA molecule, the strands in the DNA molecule increase.

Conclusion

In sum, (DNA) test is a process that helps in examining biological materials to ascertain paternity. Moreover, it also helps the forensic experts provide the evidence in cases of sexual assault since the DNA has distinct chemical properties to each person. Usually, biomaterials such as semen, bone tissues, blood, and urine are widely used in carrying out forensic investigation on suspected criminals. Apparently, the experiment showed that as the distance covered increases, the DNA size increases due to disperse convexity, showing the response of the DNA elements.

References

Bieber, F et al., 2006, “Finding Criminals through DNA of Their Relatives”, Science, vol. 312 no.1315, pp. 1315–16.

Bruce, B & Angela, V 2009, “Extracting evidence from forensic DNA analyses: future molecular biology directions”, Biotechniques, vol. 46 no 5, pp. 339-350.

Gosline, A 2005, “Will DNA Profiling Fuel Prejudice”, New Scientist, vol. 186 no. 2494, pp.12-13.

Joseph, W 1989, The Blooding, Perigord Press, New York.

Nick, W 2002, “False result fear over DNA tests The Observer”, p. 48.

Saks, M & Faigman, D 2008, “Failed forensics: how forensic science lost its way and how it might yet find it”, Annual Review of Law and Social Science, vol. 4, pp. 149–171.

Schafer, E 2008, “Ancient science and forensics”, Forensic Science, vol. 173 no. 1, pp. 40.