Laboratory Report

 

Abstract

DNA fingerprint matching using PCR is an accurate and affective way of identifying or matching DNA to an available sample. It is used widely throughout forensic science and is spoke about very highly within further research. Throughout this report the positives of PCR are discussed, along with the use of equipment and how this method is still effective, even when not accomplished completely accurately.

 

Introduction

The purpose of this experiment was to identify which suspects DNA, out of the four samples provided, matched the DNA that was given as DNA found at a scene. The DNA matching was completed using Polymerase Chain Reaction (PCR). The DNA used within this experiment was provided in a DNA fingerprinting experiment kit, created by Edvotek (EDVOTEK, 2017). However, DNA matching using PCR is frequently used by forensic professionals to determine DNA profiling of suspects using minute amounts of DNA which has been extracted from a crime scene. (Sinelnikov and Reich, 2017). Cavanaugh and Bathrick (2018) argue that PCR amplification is one of the most effective forms of DNA matching within forensic science. PCR is found to be more effective than other methods as DNA samples can be directly added to an amplification reaction, rather than being exposed to DNA extraction, purification or quantification. All of which can damage or contaminate DNA samples. This method also allows maximum amounts of DNA to be extracted, allowing for less error when matching DNA fingerprints. This is a vital advantage when working within forensic science.

Materials

  • DNA samples (provided by EDVOTEK)
  • 5 PCR tubes
  • Primer mix
  • PCR Edvobead
  • Centrifuge
  • (50x) Buffer
  • Distilled water
  • Flask
  • Microwave
  • 7x7cm casting tray – with rubber ends and well templates
  • Electrophoresis chamber
  • Timer

Method

Firstly, we began by labelling 5 PCR tubes to ensure that the experiment was completely accurate. The tubes were labelled; Crime scene (CS), suspect 1 (S1), suspect 2 (S2) suspect 3 (S3) and suspect 4 (S4). To prepare each individual PCR reaction, each tube was filled with 20 micro litres of the primer mix provided, then 5 micro litres of the crime scene DNA (for the PCR tube labelled CS) and finally one PCR Edvobead. This was repeated for each tube, but the appropriate DNA was added to the appropriately labelled tube. Each tube was then gently shaken to mix the solution inside, and to ensure that the Edvobead was fully dissolved. Next, the samples were placed inside the centrifuge to spin and separate the DNA from the rest of the solution, making it easy to collect from the bottom of the tube. The DNA was then amplified using PCR. The PCR cycle started with initial denaturation starting at 94 degrees Celsius for 3 minutes. After, the cycle continued with conditions of 94 degrees Celsius for 30 seconds, then 55 degrees Celsius for 65 seconds and then 72 degrees Celsius for 30 seconds. This cycle was repeated 30 times. Before beginning electrophoresis, 5 micro litres of 10x gel loading solution was added to each tube.

The next step was to dilute 0.5ml of concentrated (50x) buffer with 24.5ml of distilled water into a flask. 0.25g was then added to the solution to create a total volume of 25ml. The agarose powder was then dissolved in the solution by microwaving it on high for 1 minute. The flask was taken out of the microwave and swirled to see if the agarose had dissolved. The flask continued to be heated for short 15 second bursts until the liquid was completely transparent – indicating that the agarose had been dissolved. The was set aside to cool down, whilst waiting, the rubber end caps were placed at the ends of 7x7cm the gel-casting tray that the gel would be placed in. The well template, or comb, was then clipping into the tray, ready for the solution to be poured in. Once the flask containing the solution had cooled down enough so that it could be touched without gloves, the agarose solution was poured into the gel-casting tray. The gel was left for over 20 minutes to allow enough time for setting. Once the gel was completely set the rubber end caps were removed, along with the comb. The comb had to be removed extremely carefully to ensure that none of the well moulds were damaged.

The tray containing the gel was then placed inside the electrophoresis chamber. 1x electrophoresis buffer was then poured into the chamber, until the tray was completely submerged. Lane 1 of the wells was filled in with the ladder sample. Then following that each DNA sample that was prepared earlier (CS,S1,S2,S3,S4) was placed in an individual well within the gel, in that order. The safety cover was then clipped onto the chamber, and the appropriate leads were attached to the appropriate power source (Red – Red, Black – Black). The electrophoresis chamber was then turned on for approximately 55 minutes. After the electrophoresis was complete, the gel trays were removed from the chambers, and the gel was then removed from the tray. The gels were then taken to a different lab where they were scanned by a technician to produce an image of the results.

(EDVOTEK, 2017)

Results

L S4 S3 S2 S1 CS L

(Essential Laboratory Techniques : Crime Scene Gel, n.d.)

The image above is the photo that was produced after the gel had been scanned by the technician. From order of right to left, the wells read; Ladder marker, crime scene, suspect 1, suspect 2, suspect 3, suspect 4 and the final ladder marker. As shown by this image the suspect that was the closest match to the crime scene DNA was suspect 3, as indicated by the DNA highlighted within the wells.

Discussion

From the results shown in the image provided, it can be seen quite clearly that the wells at the top of the image are more intact and more obvious to see. But towards the bottom of the image it appears the wells become less distinct. As discussed previously, the agarose gel solutions were set in gel casting trays size 7x7cm, and the electrophoresis chamber was turned on for around 55 minutes. From the results shown we can see that the wells almost taper off, towards the end of the image. As shown from the image below it is quite clear that all the wells are intact and obvious to see when the gel was scanned for an image.

(Zhang et al., 2015)

This could suggest that the agarose gel became damaged during the experiment because as the DNA was migrating during the electrophoresis (DNA migrates towards the red [positive] electrode) the gel may have become damaged if the chamber was too small and if the current was too strong. If the DNA didn’t have enough room to migrate properly the larger sections of DNA could have damaged the wells whilst travelling, creating a less clear picture. With this in mind, the electrophoresis chamber may also have been left on for too long, although the manufacturer guides were followed correctly.

Although the gel may have been damaged during the experiment, it is still clear from the results which suspect DNA matched the DNA that was found at the crime scene. This gives indication that although the experiment may not have been completed 100% correctly, it still shows how accurate PCR can be, giving and indication of why it is so popular and widely used within forensic science and other sciences. (Pounder et al., 2005)

Conclusion

In conclusion, the experiment was successful in determining which suspect DNA matched that of the crime scene DNA. DNA fingerprint recognition using PCR has been proven to be extremely successful and accurate. The method produces clear imagery and evidence, even if the process of electrophoresis isn’t always correct. This shows the benefits of PCR and how determining and matching DNA fingerprints has developed.

References

Cavanaugh, S. E. and Bathrick, A. S. (2018) ‘Direct PCR amplification of forensic touch and other challenging DNA samples: A review.’ Forensic Science International: Genetics, 32(Supplement C) pp. 40–49.

EDVOTEK (2017) ‘DNA fingerprinting using PCR.’

Essential Laboratory Techniques : Crime Scene Gel (n.d.). [Online] [Accessed on 9th January 2018] http://moodle.writtle.ac.uk/mod/resource/view.php?id=105232.

Pounder, J. I., Williams, S., Hansen, D., Healy, M., Reece, K. and Woods, G. L. (2005) ‘Repetitive-Sequence-PCR-Based DNA Fingerprinting Using the DiversiLab System for Identification of Commonly Encountered Dermatophytes.’ Journal of Clinical Microbiology, 43(5) pp. 2141–2147.

Sinelnikov, A. and Reich, K. (2017) ‘Materials and methods that allow fingerprint analysis and DNA profiling from the same latent evidence.’ Forensic Science International: Genetics Supplement Series, 6(Supplement C) pp. e40–e42.

Zhang, Y., Suehiro, Y., Shindo, Y., Sakai, K., Hazama, S., Higaki, S., Sakaida, I., Oka, M. and Yamasaki, T. (2015) ‘Long-fragment DNA as a potential marker for stool-based detection of colorectal cancer.’ Oncology Letters, 9(1) pp. 454–458.

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