Molecular Genetics: Catching the Criminal Using Electrophoresis

Introduction

A sample of DNA found in a crime scene was provided along with five suspects. Their DNA was then processed using restriction enzymes and Agarose Gel Electrophoresis. The objective of this lab was to match a criminals DNA to a crime scene using restriction enzymes EcoRI and Pstl with Agarose gel electrophoresis. Restriction enzymes cut DNA at a specific base pair site recognized by the enzyme, which then turns one single strand of DNA into many fragmented strands of DNA.

EcoRI recognizes and cuts the palindromic base pair sequence GATTC while Pstl recognizes and cuts the palindromic base pair sequence CTGCAG. Agarose gel electrophoreses separates these fragmented DNA by their size. The negatively charged DNA moves through the Agarose gel to the positively charged end of the gel. The smaller fragments move through the gel more quickly allowing a linear view of the fragmented DNA when the process is complete. Since each individuals DNA will be cut into different size fragments when restriction enzymes are applied we can match one of the suspects to the crime scene DNA sample.

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This process enables an individual’s DNA to be matched, much like a fingerprint, to a sample of unknown DNA. Methods An enzyme mix of EcoRl and Pstl was added 10 microliters at a time to the crime scene sample and suspect samples one through five each containing 20 microliters of DNA. A new pipet was used for each transfer of the enzyme mix to ensure that there was no cross contamination of the suspects. To guarantee the enzyme reacts with the DNA the six samples mixed with enzyme were then centrifuged.

The samples were incubated at 37 ° C for 45 minutes, after incubation 5 microliters of dye were added to each sample. During this time an Agarose gel was cast using an 8 well comb. The Agarose gel was placed in the electrophoresis chamber with the wells at the cathode end and 275mL of electrophoresis buffer was added. In the first well 10 microliters of Hindlll DNA marker was added. This marker was provided dyed. In the following wells 20 microliters of each sample was added, Table 1 provides the lane information. The volts were set at 120 Volts and the sample was electrophoresed for 30 minutes.

After the gel was electrophoresed it was transferred into a container and dyed with Fast Blast DNA stain so the DNA fragments could become visible to the eye. Results Figure 1 below shows the samples once they have been dyed. To the naked eye it would appear that the closest match to Lane 2 (the crime scene) would be Lane 4 (Suspect 2) but to verify this conclusion you need to calculate the size of the bands. To compare the samples the size of each marker band was measured from the well to the band in mm and graphed with the given size of each band as shown in Graph 1.

In the first column of Table 2, Hindll size in base pairs was provided, to find the approximate size of the other samples the distance of each band was plugged in as an x-value to the y=-142x+13214 equation found using excel on the best fit line on Graph 1. Comparing the crime scene column to suspects one through five it was found that Suspect 3 was the criminal. His DNA fragments were of similar size and travelled a similar distance through the electrophoresis gel. Discussion- There is a pretty serious error with the calculations of size in base pairs as presented in Table 2.

Some of the base pair lengths were found to be negative numbers which does not properly correlate to the proposed size of the bands. This error was most likely to have happened in the graphing of the marker. In the results it was discussed that Suspect 3 is most likely to be the criminal but this result was found by disregarding the negative values. If the error was corrected and the correct size measurements were found the suspect found to be the criminal may have been different.

Since the values for size had an error in them the criminal could not be positively identified.

Conclusion

In this lab it was clear that pairing restriction enzymes with gel electrophoresis makes it possible to match a DNA sample to an individual. Applying the restriction enzyme cuts each DNA sequence into a unique size and amounts of fragments for each sample. This unique combination of sequences is what makes it possible to run the fragments through an electrophoresis gel that separates the fragments into a unique “fingerprint. Although a suspect was not properly identified to the crime scene sample, it is clear how it would be possible to identify a criminal. Table 1- This table lists each lane of the electrophoresis well and what sample was pipeted into it and how much of each sample in microliters. Lane one starts on the left hand of the well. Graph 1- The graph provides a scatter plot of the marker in lane 1, in a log scale, linear fit with a best fit line through it. The equation for slope found was y=-142x+13214.

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