CSI: INVESTIGATION BY DNA PROFILES AND RESTRICTION ENZYMES Introduction It is common to hear reports in the media of crimes that

have been solved with the assistance of DNA analysis (Brown, 2006). If enough blood, semen or tissue is found at a crime scene, forensic laboratories can determine the blood type or tissue type by using antibodies to detect specific cell surface proteins (Campbell et al, 2006). This method unfortunately requires large amounts of fresh samples and is not a strong source of evidence as several people have the same blood or tissue type and thus it only narrows down a few suspects (Campbell et al, 2006). DNA testing has a high level of certainty as DNA sequencing is unique for each individual except for identical twins (Campbell et al, 2006). To obtain a profile a Short Tandem Repeat (STR) needs to be generated from blood or other samples (Bustamante et al, 2007). Profiling makes use of the STRs which are short sequences, 1-13 nucleotides long that are repeated several times in tandem array. In DNA profiling, the alleles of a selected number of different STRs are determined (Brown, 2006).When restriction enzymes are used to cut STRs and the results are analysed (by electrophoresis or southern blotting a technique not investigated in this experiement), a pattern of bands are produced which is described as a ‘DNA fingerprint’ as it is unique for each individual (Cummings, 2003). DNA-modifying enzymes that cut DNA at specific sequences produce DNA segments which are called restriction endonucleases or restriction enzymes (Knox et al., 2005). The fragments that are formed when DNA is digested by restriction enzymes can be joined together in new combinations using DNA ligase to create recombinant DNA (Knox et al, 2005). DNA fragments of different sizes are separated by gel electrophoresis (Knox et al, 2005). Electrophoresis is a technique used in separating molecules in a mixture under an applied electric field (Lodish et al., 2004). The

dissolved molecules in an electric field move at a speed determined by their charge to mass ratio. Many proteins or nucleic acids have very similar charge to mass ratio and electrophoresis of these macromolecules results in little or no separation of molecules of different lengths, thus successful separation of proteins can be carried out in various gels rather than in a liquid solution (Lodish et al., 2004). In this experiment, we will only use restriction enzymes and agarose gel electrophoresis techniques for our crime investigation.

Materials and Methods Refer to Biology 1004 Molecular Biology practical manual, “Week 8, DNA Profiling Experiment” p 49 - 51 for materials and methods (see reference). Experiment was carried out as per the lab manual, with the exception in the procedure where 5µL samples of DNA were used in step 1 opposed to the outlined 2µL and a predetermined ladder was provided by demonstrator for comparison.

Aim To investigate how DNA profiling may be used to assist in solving a crime using restriction enzymes techniques and separation by gel electrophoresis.

Results The DNA samples were run through gel electrophoresis where fragments of different sizes were separated for comparison and analysis.

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Figure 1. Gel slide of DNA bands extracted from the three suspects found under UV light. The amplified fragments are visualized on agarose gel. Lane 1: DNA Ladder. Lane 2: Blank. Lane 3: Suspect X. Lane 4: Suspect Y. Lane 5: Blank. Lane 6: Suspect Z. Lane 7: Evidence sample. Lane 8: Blank.

Using the information from the gel photo (see figure 1), an initial comparison can be made of what sample matches the provided evidence. Suspect Y is observed to be closely matched with the evidence, but at this point further calculations are required to confirm such implications.

An accurate measurement of the distances between each fragment can then be taken from figure 1 as the number and sizes of the DNA bands are known and the distance travelled of each are clearly displayed.

Standard Curve for DNA Ladder
4
size of fragment (log bp)

3.5 3 2.5 2 1.5 0 5 10 15 20 25 30 35 40 45 50
distance travelled (mm)

y = -0.0652x + 4.7011

Figure 2. Standard curve for the DNA ladder of the fragments (in log measurements) in relation to the distance travelled of each band. The added trend line and its equation provides the basis for further calculations of DNA samples.

Running a sample of a DNA ladder in the gel slide also assists in the analysis. The distance travelled by each fragment was measured from figure 1 and a predetermined ladder was provided by the demonstrator to help identify the size of fragments. Using this information a standard curve for the ladder was produced (see figure 2). From this curve, a trend line was added which produced an equation which provides the basis for further calculations in the DNA samples of the suspects for this case.

The measurements of samples X, Y, Z and E were taken from the wells, and using this information and the equation provided from figure 2, an accurate fragment size could be calculated for numerical comparison.

Sample calculations: Eg for fragment 1 in sample X, Distance travelled = 30 mm Substitute into equation

Y = -0.0652x + 4.7011 = -0.0625 (30mm) + 4.7011 = 2.75 (size in natural log) Therefore for size in bp, take natural log to the power of 10. 102.75 = 556.03 bp

Using the calculations above, it is possible to further investigate the fragment sizes in each sample and evaluate whether it is probable that the evidence presented matches any of the given samples.

Analysis of Fragment Sizes in DNA Samples
2000 1800 Fragment Size (bp) 1600 1400 1200 1000 800 600 400 200 0 1 2 3 Fragment Sample 4 5 Sample X Sample Y Sample Z Evidence

Figure 3. The size of each fragment (bp) compared to other DNA samples from the three suspects.

Again, sample Y appears to be closely matched to the evidence as their lines appear to have the most similar trends as depicted in figure 3 (see appendix for further details). Although sample Z follows similar trends initially, sample Y coincides with the evidence at fragment points 4 and 5 in figure 3, setting it apart from the other samples tested.

Discussion and Conclusion Sample X displays an unconvincing relationship to the evidence in both figure 1 and 3 as the distance travelled and its fragment size are not so closely matched to the values presented in the evidence. Sample Z appears closely matched to the evidence sample in both figures 1 and 3, but the fragment size that is presented after measurements and calculations appears to be slightly larger than that of the evidence. Nevertheless, this sample still represents a likely suspect involved at the crime scene. Sample Y showed similar trends in Figures 1 and 3 to the evidence provided for this case and further comparison across groups in the class confirms the trends found across all samples with sample Y being the closest match to the evidence. Irregularities found in the results can be attributed to crude measuring techniques. Measurements were carried out in millimetres, whereas nanometres for instance would have given a more precise account of the distance travelled by the DNA bands. Furthermore, this experiment was dependant on accurate methodologies especially in loading and running the agarose gel. Errors to this process (or other steps in the method) would cause an unclear scan on the agaraose gel making a proper analysis of the DNA fragments difficult. At a crime scene, the biological evidence found, such as blood, semen hairs etc, would be unintentionally left behind and the quantities would be very small. DNA fingerprinting might be used in such cases with the assistance of polymerase chain reaction (PCR), where the STRs are amplified before testing, thus the problem of minute quantities can be overcome (Cummings, 2003). In criminal cases, analysis of DNA fingerprints requires more than a test through agarose gel, rather a combination of probability theory, statistics and population genetics to estimate how frequently a particular fingerprint might be found in an

individual or in the general population (Cummings, 2003). Suggestions for future experiments would be to investigate this population frequency for STR alleles as it evaluates the chances of another likely candidate within a population of having the exact genetic information. It would not prove an individual’s guilt but rather strengthen a case, along with other DNA evidence, relating to a person’s involvement in a crime. In conclusion the sample that most closely matches the evidence presented is that of Sample Y, the boyfriend of the deceased. This is due the similarities in fragment size and trends calculated from this analysis. It should be noted that the presence of this individuals DNA at the scene of the crime does not necessarily imply that he was involved in the murder, but rather his presence at that location at some point in time.

References Bustamante, I.T., Mata, F.S., et al (2007) Application of chemometric tools for automatic classification and profile extraction of DNA samples in forensic tasks, Analytica Chimica Acta, 595, pp 43-50.

Brown, T.A. (2006) Gene cloning and DNA analysis: An Introduction, pp 346-350. Blackwell Publishing, Oxford, UK. Campbell N, Reece J. & Meyers N. Biology, 7th Edition, pp 402-406. Pearson Education, Australia 2006. Cummings, M.R (2003) Human Heredity: Principles and Issues, 6th Edition. Pp 323325. Brooks/Cole, Pacific Grove, CA. Knox, et al. (2005) Biology: an Australian focus, 3rd edition, pp 54-57. McGraw Hill, North Ryde, Australia. Lodish, H., Berk, A., et al. (2004) Molecular Cell Biology, pp 87-89, W.H Freeman and company, New York.

Appendix Calculations of fragments relating to figure 3.
Sample X Fragment 1 Fragment 2 Fragment 3 Fragment 4 Distance trav (mm) 30 31 33 35 Nat. Log 2.75 2.68 2.55 2.42 Size (bp) 556.03 478.52 354.41 262.48

Sample Y Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5

Distance trav (mm) 22 23.5 25 28 35

Nat. Log 3.27 3.17 3.07 2.88 2.42 Nat. Log 3.2 3.07 2.94 2.75 2.68 Nat. Log 3.23 3.14 3.01 2.88 2.42

Size (bp) 1847.99 1475.37 1177.88 750.76 262.48 Size (bp) 1590.38 1177.88 872.37 556.03 478.52 Size (bp) 1714.35 1368.67 1013.68 750.76 262.48

Sample Z Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5

Distance trav (mm) 23 25 27 30 31

Sample E Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5

Distance trav (mm) 22.5 24 26 28 35

Appendix questions (see attached)

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