DNA fingerprinting lab Colin and Brianna

Abstract
The widespread use of electrophoresis has played an essential part in mapping the human genome. The process of electrophoresis was developed in the mid-1960s and enhanced in the early-1970s. DNA fingerprinting is now used in many different areas of research including forensics and the study of bacterial epidemics. This lab is meant to show the process of DNA fingerprinting through electrophoresis by simulating a crime scene using bacterial plasmids and restriction enzymes Eco-R1 and Pst1. The bacterial plasmids were used over human plasmids for simplicities sake. The experiment was set up according to standard procedure for an agarose gel electrophoresis. The study showed how DNA fingerprinting helps find similarities in cleaving sites between different plasmid samples. The results were analyzed based on plasmid maps and the observed agarose gel. The observed DNA fragments were compared to the expected fragments and some discrepancies were found. These could be due to errors in preparation method of the agarose gel. The benefits of DNA fingerprinting stretch over all areas of science and help researchers discover new aspects of bacterial and human genomes.

Background
The widespread use of electrophoresis has played an essential part in mapping the human genome (Vesterberg). The discovery of the different parts of the genome has benefited many areas of investigative science. In the early 1980’s, Alec Jefferies began the revolution of DNA fingerprinting and published his method in the 1985 Nature journal article, Hypervariable ‘minisatellite’ regions in Human DNA (Jobling 740, see attached timeline). He enlightened the world of genetics by discovering minisatellites, sections of DNA containing a small sequence of bases, helped determine multi-band patterns commonly known as “fingerprints” (Jobling and Gill 740). DNA fingerprinting revolutionized forensics as a whole. It helps solve burglaries, murders, crime scenes, and even helps confirm paternity tests.
At the Institute of Virology in Glasgow in the mid-1960s, Vin Thorne wanted to find a better way to classify the DNA forms that he was studying from purified polyomavirus. Through his knowledge of frictional and electrical forces, he was able to use electrophoresis with agar to separate polyomavirus DNA fragments, but only after the segments expressed radioactive traits. In the early-1970s, however the use of restriction enzymes greatly increased the popularity of Thorne’s method because the DNA could be detected even if it was not radioactive. Restriction enzymes are used to cut DNA into small sequences. Eco-R1 and Pst1 are both commonly used enzymes that cut between the A and G bases. The Eco-R1 enzyme only cuts when the sequence 5’ GAATTC 3’ is present and the Pst1 cuts the DNA at the sequence 5’ CTGCAG 3’ (National Library of Medicine, Hartl and Jones 433).
The method that is used today, however, came from a group of investigators working at Cold Spring Harbor Laboratory in the early-1970s. The investigators discovered that the fragments of DNA were more visible when the agar was dyed (Sambrook and Russell). Since the movement of the DNA fragments was unaffected by the dye the use of the dye became common practice and allowed researchers to insert photographs of the gel plates into computer databases.
This lab simulates how forensic analysts would perform Agarose Gel Electrophoresis with DNA samples from the crime scene and multiple suspects with one suspect’s DNA fingerprint matching the crime scene’s DNA fingerprints. This simulated crime scene uses bacterial plasmids instead of human plasmids. Bacterial plasmids are used instead of human plasmids because, in the educational laboratory setting, human plasmids, with 3 x 109 base pairs, are too large. The bacterial plasmids used are significantly easier to work with because they are smaller, with the maximum number being 9,481 base pairs (Hartl and Jones 222-223).

Materials and Methods
A 50-mL sample of 1% weight to volume agarose solution was made in TAE buffer by the method previously described (Sambrook and Russell). While the agarose was setting, 15 microliters of the EcoR1 and Pst1 restriction enzyme mix was added to each DNA sample then the DNA was incubated at 37° C with air flow for 30 minutes. When the DNA was finished incubating 5 microliters of dye was placed into each vial of DNA. The electrophoresis was then set up according to Sambrook and Russell.

Results
After electrophoresis was completed, the agarose gel showed the various DNA sequences from the crime scene along with plasmid sequences T1 through T5 from the simulated crime scene, followed by a ladder sequence. The crime scene sequence had three DNA fragments close together, followed by another fragment much further down. T1 had three DNA fragments which were spaced evenly apart, while T2 had three DNA fragments as well. The T2 showed similar spacing to the last two fragments, whereas the first fragment was spaced further apart, towards the top. T3 had a total of four fragments with three evenly spaced apart and one further down. Unevenly spaced, there were three T4 fragments. In T5, there were four unevenly spaced fragments, starting half way down the gel. T5 was then followed by a ladder that started at the top end of the gel and contained three evenly spaced DNA fragments.

Discussion
There are many different types of electrophoresis that are used for a cornucopia of research topics. One more widely used method is Pulsed-field Agarose Gel Electrophoresis (PFGE). PFGE is a method that is able to produce larger pieces of DNA fingerprints (Joppa et. Al). The benefits of the larger fingerprints include bacteriophage typing and serotyping to determine the risk different bacteria are for causing an outbreak and also more accurate forensic DNA results (Tenover et. al 2233). In true-life forensic situations analysts need the suspect’s DNA fingerprints to match the crime scene fingerprints perfectly. This is also the case in this experiment. So, the DNA taken from the crime scene will match that of at least one suspect’s DNA sample.
In this experiment, each bacterial plasmid has specific fingerprints based on the plasmid maps (Bio-Rad). However, the observed results differed slightly from what was expected. The different bacterial plasmids in the agar were missing some expected fragments. This shows that the restriction enzymes did not cleave the plasmid DNA at the proper places. For T1 and T2 the expected result was to have 4 DNA fragments but the observed results only showed 3. For T4, the expected result was to have 4 DNA fragments but only 3 were observed and T5 had the largest discrepancy with the expected results being 8 and the observed result being 4. However, the hypothesis was supported because the observed results from the CS and T3 plasmids matched and had the expected number of DNA fragments. The errors in the plasmid DNA fragments could have occurred because when the wells in the agarose were filled with the DNA, they were over-filled so some DNA mixed.
The experiment used bacterial genomes because they have a much smaller number of base pairs than human genomes, making them easier with which to work. Also, by using the bacterial genomes the base pair sequence is known and therefore the location of each cleavage site is known. If human genomes were used, it would be impossible to tell where the cleavage sites would be located. Since the human genomes have so many base pairs, the chance of mutation is greater, making it virtually impossible to work with in an educational setting (Bio-Rad).

Works Cited

Bio-Rad Laboratories. Appendix E. 2008.
Hartl, Daniel L, and Elizabeth W Jones. Genetics: Analysis of Genes and Genomes. Mississauga: Jones and Bartlett, 2009.
Jeffreys, Alec J, Victoria Wilson, and Swee Lay Thein. “Hypervariable ‘minisatellite’ regions in human DNA .” Nature 314 (Mar. 1985): 67-73.
Jobling, Mark A, and Peter Gill. “Encoded Evidence: DNA in forensic analysis.” Genetics 5 (Oct. 2004): 739-751.
Joppa, Barbara, et al. “Pulsed Field Electrophoresis for Separation of Large DNA.” Probe 2.3 (1992).
National Library of Medicine. “MeSH Descriptor Data.” National Library of Medicine. 2008. 21 Jan. 2009 <http://www.nlm.nih.gov>.
Sambrook and Russell. Molecular Cloning: A Lab Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2001.
Tenover, Fred C, et al. “Interpreting Chromosomal DNA Restriction Patterns Produced by Pulsed-Field Gel Electrophoresis: Criteria for Bacterial Strain Typing.” Clinical Microbiology 33.9 (1995): 2233-2239.
Vesterberg, O. “A short history of electrophoretic.” Electrophoresis 12 (Dec. 1993):

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