DNA Fingerprinting Lab
Rhode Island State Crime Lab
Providence, RI 02903
Rhode Island District Court
Providence, RI 02903
Dear District Attorney,
Enclosed is a report of the results of DNA fingerprinting techniques for a gang shooting that occurred on September 1, 2003 at about 11:30 P.M. in South Providence, Rhode Island, in which two people were seriously injured and another was killed.
Analysis of the results of the DNA samples from five suspects as well as the back steps of the house where the crime took place indicates that suspect number three was at the scene while the shooting occurred.
This report details the procedures used to obtain and analyze the DNA samples, including variables that could possibly have interfered with the results.
Biochemistry Lab Technician
September 25, 2003
The Theory of RFLP, or Restriction Fragment Length Polymorphism, and aragose gel electrophoresis were used to determine the results of the DNA fingerprinting test. The DNA from five different suspects, as well as the DNA from the crime scene and a pre-made HindIII lambda DNA sample, was tested. The lambda DNA was run with the samples and measured to obtain a standard. It was compared to one made in the lab to ensure accuracy of the procedure. The agarose gel provided DNA banding patterns which were used to match the crime scene DNA with the suspect at the scene. Using a Ultraviolet Light Transilluminator, the bands and their base pairs could be visually compared. Of the five suspects, number three’s DNA seems to be consistent with that at the crime scene, having the same six bands and very similar numbers of base pairs for each band, placing that person at the attack site when the crime occurred. No other suspect had the same number of bands as the crime scene DNA, which eliminates them from being present at the crime scene based on the DNA fingerprinting results alone.
A) Explanation of Theory of RFLP: Restriction Fragment Length Polymorphism
B) Explanation of Theory of Agarose Gel Electrophoresis
C) Detailed Procedure
1) Photograph of the Agarose Gel Showing DNA bands
2) Table of Data Corresponding to the Gel
E) Issues/ Concerns
Theory of RFLP:
Restriction Fragment Length Polymorphism
The Theory of Restriction Fragment Length Polymorphism, or RFLP, maps the genes that are responsible for inherited diseases. The restriction endonucleases hydrolyze DNA (Boyer 141). An individual DNA “fingerprint” can be established using RFLPs by the use of various restriction enzymes. These “fingerprints” can be made using only small amounts of DNA. This works because alleles have a variety of sites for endonuclease cleavage. Therefore, when different endonucleases are used, the allele is cleaved in different sites producing an assortment of arrays of fragments (Mathews et al. 977).
The different restriction enzymes used in this procedure were EcoRI, PstI, and HindIII. These enzymes are named for the bacteria and strain from which they are derived. EcoRI is isolated from an E. coli strain that carries a cloned EcoRI gene from E. coli RY13 (New England 39). PstI is a restriction enzyme that comes from an E. coli strain that carries the cloned PstI gene from Povidencia stuartii (New England 55). HindIII comes from an E. coli strain that carries the cloned HindIII gene from Haemophilia influenzae (New England 42). These different enzymes cleave the DNA in various places (Mathews et al 931). RFLPs are used to reveal unique DNA sequences of different individuals, such as the five suspects.
Mathews, Christopher K., K. E. van Holde, and Kevin G. Ahern. Biochemistry. 3rd ed.
Addison Wesley Longman, Inc.: San Francisco, 2000.
New England Biolabs 2002-2003 Catalog. New England Biolabs, Inc.: 2002.
Boyer, Rodney F. Modern Experimental Biochemistry. 2nd ed. The
Benjamin/Cummings Publishing Company, Inc.: Redwood, 1993.
Theory of Agarose Gel Electrophoresis
Gel electrophoresis is a process that separates macromolecules on the basis of size or change. Molecules are forced across a gel by means of an electric current provided by electrodes at each end of the electrophoresis chamber. The movement of the molecules depends upon the electric current applied, the matrix of the gel, and the size, shape, charge, and chemical composition of the molecules (Boyer 111). Agarose is a product extracted from seaweed. Agarose gels can be processed fairly quickly and are used mainly for analysis of larger nucleic acid fragments, that is, those with 200 to 50, 000 base pairs (Boyer 122).
Boyer, Rodney. Modern Experimental Biochemistry. 3rd ed. Addison Wesley Longman,
Inc.: San Francisco, 2000.
National Center for Biotechnology Information. 2 Sept. 2003 <www.ncbi.nlm.nih.gov>.
One sample from the crime scene was obtained while samples from five different subjects were collected. Ten micro-liters of each of the suspects’ DNA, all of which had unknown concentration, were placed in Epindorph tubes and mixed with ten micro-liters of EcoRI/PstI restriction enzyme mixture. One of the technicians performing the same procedure took one hundred micro-liters of DNA form samples one and three, which corresponds to the suspect numbers. She removed ninety micro-liters from each but disposed of this excess. This affected my sampling. I used the original sample tubes for samples one and three because I would probably have not gotten ten micro-liters otherwise. The samples were spun and incubated at thirty-seven degrees Celsius for approximately forty-five minutes. Three Lambda DNA samples were made. All contained four micro-liters of DNA, six micro-liters restriction buffer 2X, and one micro-liter of an enzyme, that is, one with EcoRI, one with PstI, and one with HindIII. These were also spun and incubated at the same temperature. Another technician and I made a one percent agarose gel out of 120 ml of 1XTEA and 1.2 g agarose. All samples were spun down in a microcentrifuge and frozen at -20 degrees Celsius. The liquid gel was poured and later immersed in 1XTEA after the solution gelled to keep the gel moist.
Five micro-liters of dye was added to the crime scene and suspect samples. Then, ten micro-liters of each were loaded in separate chambers in the gel. Two micro-liters was added to the lambda DNA samples and eight micro-liters of each was loaded. One hundred-ten volts was put through the gel to make the bands. The patterns formed on the gel indicated which samples were and were not consistent with the crime scene DNA. A picture of the gel was taken in an Ultraviolet Light Transilluminator, which then transferred the picture into a computer program so the patterns and base pairs could be examined. To analyze, a data table was obtained which provides the number of base pairs for each band.
Suspect number three can be placed at the crime scene according to the RFLP and agarose gel electrophoresis results. This person’s DNA gel pattern matched that found at the crime scene. Those of suspects one, two, four, and five did not match the crime scene DNA, and can, therefore be eliminated as suspects based on these procedures alone. A photograph of the agarose gel that makes apparent the DNA banding patterns of the individuals and a table that shows the number of base pairs in each band provide these results in greater detail.
The DNA was incubated in a water bath with DNA from other technicians' cases, but the caps on the centrifuge tubes were securely closed. In addition, air was trapped in the pipettor when loaded the dye into the samples, and a back-up power supply was used because the initial one stated an error. One technician in the lab affected my sampling by decreasing the amount of DNA I was able to use for samples one and three, which did not appear to but may have tampered with my results.
However, little error must have occurred due to the nearly identical banding patterns of sample three to the crime scene and extreme difference between sample one and the crime scene. Another technician ran the same data and received the same results, which can be seen in the DNA banding patterns in the photograph of the gel.
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