Who Done It? DNA Fingerprinting and Forensics *
AbstractDo you like reading detective stories or watching crime shows on television? This advanced project idea in the area of forensic science might be right for you.
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?
Andrew Olson, PhD, Science Buddies
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Last edit date: 2017-07-28
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, as shown in Figure 3, below (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.
Figure 3. DNA "fingerprints" that are being visualized by staining the gel (instead of using radioactively-labeled probes). (Photo courtesy of Bio-Rad Laboratories, Inc.)
Terms and Concepts
To do this project, you should do research that enables you to understand the following terms and concepts:
- 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
- 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?
- You can run a "virtual" DNA fingerprinting experiment at this webpage:
- Groleau, R. (n.d.). Create a DNA Fingerprint. NOVA Online: "The Killer's Trail." Retrieved May 14, 2016 from http://www.pbs.org/wgbh/nova/sheppard/analyze.html.
- For an introduction to DNA fingerprinting, see:
- Wikipedia Contributors. (2016, May 3). DNA profiling. Wikipedia: The Free Encyclopedia. Retrieved May 14, 2016 from https://en.wikipedia.org/w/index.php?title=DNA_profiling&oldid=718423054.
- GeneEd. (n.d.). DNA Forensics: The application of genetic testing for legal purposes. Retrieved May 14, 2016 from https://geneed.nlm.nih.gov/topic_subtopic.php?tid=37&sid=38
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Materials and Equipment
To do this experiment you will need the following materials and equipment:
- Forensic DNA Fingerprinting Kit from Bio-Rad (1).
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. These are available from an online supplier such as Carolina Biological catalog #215589. The minimum order size through the Carolina Biological Supply Company is a package of 25 trays.
- Water bath
- Hot plate, Bunsen burner, or microwave oven
- 250 ml flask. This is available from an online supplier such as Carolina Biological catalog #726690.
- Pipet pump
- Hot gloves
- Marking pens
- Distilled or deionized water
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Who Done It? DNA Fingerprinting and Forensics
- 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.
- 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.
- Follow the instructions in the kit to perform the DNA fingerprinting.
- Your display board should explain how DNA fingerprinting works. Take a photograph of your gel to include on your display board.
If you like this project, you might enjoy exploring these related careers:
Forensic Science TechnicianGuilty or not guilty? The fate of the accused in court lies with the evidence gathered at the crime scene. The job of the forensic science technician is to gather evidence and use scientific principles and techniques to make sense of it. It can be a grueling and graphic job, but very rewarding. If you like the idea of using science to help deliver justice, then you should investigate this career. Read more
Cytogenetic TechnologistI have black hair, you have blonde hair. I have blue eyes, you have brown eyes. These, and other characteristics, describe what we look like, how tall we are, and even what our personality is, and they are all controlled by our chromosomes. Chromosomes are packages within each of our cells that hold our genes. Our chromosomes also determine if we might inherit any genetic diseases or if birth defects are present. Extracting, testing, and examining the chromosomes from cells is the job of the cytogenetic technologist. Cytogenetic technologists work with physicians to help diagnose and treat diseases and understand human development. This is a career in which you know you will be helping someone every single day. Read more
Genetic CounselorMany decisions regarding a person's health depend on knowing the patient's genetic risk of having a disease. Genetic counselors help assess those risks, explain them to patients, and counsel individuals and families about their options. Read more
Biological TechnicianWhat do the sequencing of the human genome, the annual production of millions of units of life-saving vaccines, and the creation of new drought-tolerant rice varieties have in common? They were all accomplished through the hard work of biological technicians. Scientists may come up with the overarching plans, but the day-to-day labor behind biotechnology advances is often the work of skilled biological technicians. Read more
- 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.
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