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Project Summary

Difficulty	8  –  10
Time required	Average (about one week)
Prerequisites	High School Biology
Material Availability	This project requires you to order specialty items and have access to some laboratory equipment, see the Materials & Equipment section for more details.
Cost	High ($100 - $150)
Safety	No issues

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Sponsor

Sponsored by generous grants from Bio-Rad and its Biotechnology Explorer program

Inspiring minds want to know. Unravel the mysteries of biotech

explorer.bio-rad.com

Abstract

Objective

Can DNA fingerprinting with the help of restriction enzymes help solve a crime scene mystery? If there are several suspects, how can the different DNAs be matched to that of the crime scene DNA?

Introduction

DNA fingerprinting (also known as DNA profile analysis and DNA typing), is a method of distinguishing between individuals by analyzing patterns in their DNA. This project focuses on the first method of DNA fingerprinting to be developed, by Sir Alec Jeffreys at the University of Leicester in 1985 (Wikipedia contributors, 2006).

Jeffreys discovered regions of DNA—that he named microsatellites—that are highly variable between individuals. Microsatellites occur in "non-coding" DNA, that is, DNA that is not transcribed into messenger RNA to code for proteins. Random variations in non-coding DNA accumulate more rapidly than in coding DNA. This is because variations in coding DNA are more likely to have negative effects, since they can cause changes in the amino acid sequence of proteins, leading to changes in protein function. While some regions of non-coding DNA are involved in regulation of gene expression, many non-coding regions have no known function, and are sometimes referred to as "junk DNA." Microsatellites are one example of such "junk DNA."

Microsatellites contain variable numbers of short, repeated sequences of DNA at a specific, identifiable locus (place) in the DNA sequence. The number of repeats is highly variable between individuals.

So how is microsatellite DNA used to distinguish between individuals? The DNA sample is first cut into smaller pieces using one or more restriction enzymes. Restriction enzymes recognize specific DNA sequences and cut the DNA molecule at or near this recognition site. Because microsatellite length varies between individuals, the resulting DNA fragments will have different lengths (the technical terms is restriction fragment length polymorphisms, or RFLPs).

In order to visualize the different fragment lengths (RFLPs), the cut DNA is loaded into a slab of agarose gel in a salt solution. An electric current is applied, which causes the DNA (which is negatively charged at neutral pH) to migrate through the gel. The gel acts to separate the DNA fragments by size, since shorter fragments move faster than larger fragments through the cross-linked structure of the gel. Figure 1 shows a simple example of cut DNA fragments separated by gel electrophoresis. An actual DNA fingerprint would contain many more bands (see Figure 2, below).

Figure 1. Simplified example of DNA fragments cut by a restriction enzyme (Eco R1) and separated by gel electrophoresis. Sample A has a shorter repeated sequence (4 repeats, 40 base pairs total), while sample B has a repeated sequence nearly twice as long (7 repeasts, 70 base pairs total). At right, the resulting fragments are visualized after being run on a gel. The shorter fragment in column A has run further down the gel than the longer fragment in column B. Column M is a set of DNA fragment size standards, with fragments of known length (Rosner and Smith, 2004).

Next, the pattern of the DNA bands containing the microsatellites is visualized. This is done by denaturing the DNA in the gel (separating the double strands into single strands) and transferring it to a nitrocellulose or nylon membrane. In this transfer process, the DNA retains the banding pattern generated in the gel, with the fast-moving, smaller fragments toward the bottom and the slow-moving, larger fragments toward the top. To detect the microsatellite-containing fragments, radioactively labeled "probes" (short sequences of DNA complementary to the microsatellite sequence) are loaded over the membrane, and then washed off. Under the proper conditions, the probes will only bind to the matching (complementary) DNA sequence, so the probes specifically label the microsatellite-containing fragments.

The membrane is then placed next to a piece of x-ray film. The emissions from the radioactive probes react with silver grains in the film, so that when it is developed, the location of the microsatellite-containing bands from the gel can be seen on the film (see Figure 2). The NOVA webpage in the Bibliography (Groleau, 2000) has a "virtual" DNA fingerprinting experiment that you can run in your web browser, which is a fun way to get an overview of the procedure.

Figure 2. DNA "fingerprints" from six individuals (Rosner and Smith, 2004).

The experimental procedure in the recommended kit is similar to the one just described. You will be digesting DNA samples from two "suspects" with restriction enzymes (non-human DNA is actually used in the kit). The DNA fragments for each "suspect" are then separated by gel electrophoresis and directly stained and visualized in the gel itself (no radioactive probes necessary!) You will use the banding pattern of the fragments generated by the restriction enzymes to match "suspect" DNA to "crime scene" DNA.

Terms, Concepts and Questions to Start Background Research

To do this project, you should do research that enables you to understand the following terms and concepts:

• DNA,
• polymorphism,
• DNA fingerprinting (also called DNA profile analysis and DNA typing),
• restriction enzyme,
• gel electrophoresis,
• restriction fragment length polymorphism (RFLP).

More advanced students may also want to study the probability statistics of DNA matching as well as more recent methods used for DNA fingerprinting:

• allele multiplication rule (establishing statistical significance of DNA profile results),
• polymerase chain reaction (PCR, used for amplifying small amounts of DNA),
• short tandem repeats (STRs),
• capillary electrophoresis.

Questions

• What are some potential sources for error in crime scene DNA analysis?

For more advanced students:

• What are some of the drawbacks of RFLP-based DNA fingerprinting?
• What makes newer DNA fingerprinting techniques, such as STR analysis, more reliable than RFLP-based fingerprinting?

Bibliography

• You can run a "virtual" DNA fingerprinting experiment at this webpage:
  
  • Groleau, R., 2000. "Create a DNA Fingerprint," NOVA Online, Killer's Trail. [accessed July 5, 2006] http://www.pbs.org/wgbh/nova/sheppard/analyze.html.
• For an introduction to DNA fingerprinting, see:
  
  • Wikipedia contributors, 2006. "Genetic Fingerprinting," Wikipedia, The Free Encyclopedia. [accessed July 5, 2006] http://en.wikipedia.org/w/index.php?title=Genetic_fingerprinting&oldid=62215381.
  • Rosner, D. and C. Smith, 2004. "How Does Genetic (DNA) Fingerprinting Work?" The Naked Scientists. [accessed July 5, 2006] http://www.thenakedscientists.com/HTML/Columnists/dalyacolumn8.htm.
• This article goes through a DNA fingerprinting example (based on the popular detective board game, "Clue") and includes statistical analysis of the results:
  Porter, S.G., date unknown. "A DNA Fingerprinting Exercise for Any Type of Class," Geospiza. [accessed July 5, 2006] http://www.geospiza.com/education/docs/DNA_fingerprinting.pdf.
• Q & A with a DNA forensics expert, includes information on statistics of DNA fingerprinting:
  Henahan, S., 1995. "An Interview with DNA Forensics Authority Dr. Bruce Weir," Access Excellence, The National Health Museum. [accessed July 5, 2006] http://www.accessexcellence.org/WN/NM/interview_dr_bruce_weir.html.

Materials and Equipment

To do this experiment you will need the following materials and equipment:

• Forensic DNA Fingerprinting Kit (1), can be purchased from Bio-Rad Laboratories http://www.bio-rad.com, catalog #166-0007EDU.
  Note: Bio-Rad Kits are sold directly to schools. To purchase, please have your school contact Bio-Rad at 800-424-6723 to verify account information and to place the order for you. Existing accounts will have orders processed within a day, and establishing an account will take approximately 48 hours.

In addition to the kit, you will also need access to the following laboratory equipment:

• Automatic micropipets with tips
• Gel electrophoresis equipment
• Gel illuminator (diffuse white light source)
• Camera for photographing gel
• Gel staining tray
• Water bath
• Hot plate, Bunsen burner, or microwave oven
• 250 ml flasks
• Pipet pump
• Hot gloves
• Marking pens
• Distilled or deionized water
• Ice

Experimental Procedure

1. Do your background research so that you are knowledgeable about the terms, concepts, questions above. The NOVA webpage in the Bibliography (Groleau, 2000) has a "virtual" DNA fingerprinting experiment that you can run in your web browser, which is a fun way to get an overview of the procedure.
2. Before starting the experiment, read through all of the instructions in the Bio-Rad kit, and make sure that you have access to all of the necessary laboratory equipment. Note: If your school doesn't have the necessary equipment try contacting a local college or biotechnology company and asking if you could do your experiment there.
3. Follow the instructions in the kit to perform the DNA fingerprinting.
4. Your display board should explain how DNA fingerprinting works. Take a photograph of your gel to include on your display board.

Variations

• More advanced students should study the statistical methods for calculating probabilities of making an erroneous match using increasing numbers of identified loci (see Porter, date unknown).
• Another advanced project idea would be to learn about PCR-based DNA profile analysis techniques and to design an experiment using these techniques. PCR-based kits are also available.

• For more science project ideas in this area of science, see Biotechnology Project Ideas.

Credits

Andrew Olson, Ph.D., Science Buddies

Last edit date: 2009-03-06 12:00:00

Career Focus

If you like this project, you might enjoy exploring careers in Biotechnology.

	Biochemist Growing, aging, digesting—all of these are examples of chemical processes performed by living organisms. Biochemists study how these types of chemical actions happen in cells and tissues, and monitor what effects new substances, like food additives and medicines, have on living organisms.			Agricultural Technician As the world's population grows larger, it is important to improve the quality and yield of food crops and animal food sources. Agricultural technicians work in the forefront of this very important research area by helping scientists conduct novel experiments. If you would like to combine technology with the desire to see things grow, then read further to learn more about this exciting career.

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