Abstract
Do you like solving mysteries? In this experiment, you can find out how a DNA fingerprint can help you figure out whodunit. The answer might just be in the "sequence" of events!Summary
Objective
In this experiment you will test if unique DNA sequences can create individual fingerprints that are also unique.Introduction
All living things come with a set of instructions stored in their DNA, short for deoxyribonucleic acid. Whether you are a human, rat, tomato, or bacteria, each cell will have DNA inside of it. DNA is the blueprint for everything that happens inside the cell of an organism, and each cell has an entire copy of the same set of instructions. The entire set of instructions is called the genome and the information is stored in a code of nucleotides (A, T, C, and G) called bases. Figure 1 shows an example of a DNA sequence that is 12 base pairs long:
Figure 1. DNA Sequence.
Every individual has its own DNA code, but how can a code with only four letters be unique? It is hard to imagine how a code with so few parts can hold so much information. The key is that the longer the code is, the more unique sequences there can be. Table 1 is showing how many unique sequences are possible for a piece of DNA of a certain length in base pairs (bp):
| DNA Length (bp) | How Many Unique DNA Sequences are Possible? |
|---|---|
| 1 bp | 4 = 4 |
| 2 bp | 4 × 4 = 16 |
| 3 bp | 4 × 4 × 4 = 64 |
| 4 bp | 4 × 4 × 4 × 4 = 256 |
| 5 bp | 4 × 4 × 4 × 4 × 4 = 1,024 |
| 6 bp | 4 × 4 × 4 × 4 × 4 × 4 = 4,096 |
| 7 bp | 4 × 4 × 4 × 4 × 4 × 4 × 4 = 16,384 |
| 8 bp | 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 = 65,536 |
| 9 bp | 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 = 262,144 |
| 10 bp | 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 × 4 = 1,048,576 |
For example, a two base pair long DNA sequence can be one of sixteen different sequences: AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, or GG. Longer sequences have even more possibilities. The 12 base pair sequence shown above is only one of 16,777,216 different DNA sequences that are possible for a piece of DNA that size! Considering that the entire human genome is 3 billion DNA bases long, the number of possible combinations is practically infinite. But the truth is, most of the DNA from person to person is the same. Because we are of the same species, our DNA is about 99.9% identical to each other. Even the DNA of a chimpanzee is 99% identical to our DNA.
With all of the similarities in the DNA sequences of humans, why does DNA fingerprinting still work? In this experiment you will investigate whether or not unique DNA sequences will generate unique DNA fingerprints. You will use an online random sequence generator to "make" pieces of DNA. Then you will use another online program to make a DNA fingerprint of each piece of randomly generated DNA. Will fingerprints with different DNA sequences look different or the same?
Terms and Concepts
To do this type of experiment you should know what the following terms mean. Have an adult help you search the Internet, or take you to your local library to find out more!
- DNA sequence
- Genome
- Nucleotides (A, T, C, G)
- Base pairs
- Restriction enzyme
- DNA gel
- DNA fingerprinting
Questions
- What does a DNA sequence look like?
- What does a DNA fingerprint look like?
- What makes DNA fingerprints look unique? Is it the DNA sequence?
Bibliography
Here are two great tutorials to learn about DNA fingerprinting and its applications:- National Academy of Sciences. (2007). Putting DNA to Work - DNA and Criminal Justice - How DNA Determines Guilt or Innocence Marian Koshland Science Museum, National Academy of Sciences (NAS), Washington, D.C. Retrieved April 12, 2013.
- Cold Spring Harbor Laboratory. (2003). DNA Interactive: Applications. Dolan DNA Learning Center, Cold Spring Harbor Laboratory, NY. Retrieved March 6, 2007.
- Maduro, M. (n.d.) Random DNA Sequence Generator. Department of Biology, University of California, Riverside. Retrieved March 6, 2007.
- UMass Medical School (2015). BioTools @ UMass Medical School. Retrieved October 8, 2015.
- Kimball, J. (2003). Restriction Enzymes. Andover, MA: Kimball's Biology Pages. Retrieved March 6, 2007.
- Wikipedia contributors. (2013, April 3). Restriction enzyme. Wikipedia, The Free Encyclopedia. Retrieved April 12, 2013.
Materials and Equipment
- A computer with Internet connection
- Java-based web browser
- Lab notebook and pencil
- Printer
- Scissors
- Glue
Experimental Procedure
- The first step is to make a piece of DNA using the Random DNA Sequence Generator shown in Figure 2.
- Enter "1000" in the box for the Size of DNA in bp, and leave the setting for the GC content at 0.50 (which will give you half G+C and half A+T).
- Click the generate button and you will get a random piece of DNA shown in the text box:
A random DNA sequence generator hosted on ucr.edu. This generator allows you to set variables such as the number of base pairs and how often G and C will show up in the end result.
Figure 2. Screenshot of the Random DNA Sequence Generator tool. - Print this page, cut it out, and paste it into your lab notebook for your records. Choose a name for this piece of DNA and write the name in your lab notebook ("Suspect #1" for example).
- Double-click in the text box to select your DNA sequence, then copy it to the clipboard by selecting "Edit" and then "Copy" from your file menu.
- The next step is to "Cut" your piece of DNA and run it through a gel matrix to make a fingerprint by using this Restriction Analyzer tool.
- The site has several sections. In the top section that says "DNA SEQUENCE INPUT & SETTINGS," shown in Figure 3, click inside the text box and paste your DNA sequence from the clipboard by selecting "Edit" and "Paste" from the file menu. Leave all of the settings to the default settings. Then click the "Apply & Analyze" button.
umassmed.edu hosts a TCAG restriction mapping program that creates a report based on DNA sequences that you upload or paste into the box.
Figure 3. Screenshot of the "DNA SEQUENCE INPUT & SETTINGS" section where you enter your generated DNA sequence.
- Once you have clicked the "Apply & Analyze" button, scroll down to the next section, "RESTRICTION SITES OVERVIEW" (Figure 4). This sections shows you all the different restriction sites that are present at least one time (left), are absent (middle), or are present only one time (right) within the DNA sequence you are investigating.
SubSequence Selection and Numbering Options in the TCAG restriction mapping program. Default options are selected under Sequence should be analyzed from bases (end), and the sequence should be analyzed as (linear).
Figure 4. Screenshot of the "RESTRICTION SITES OVERVIEW" section.
- Scroll down to the next section of the Restriction Analyzer tool that is titled "RESTRICTION FRAGMENTS" (Figure 5). Keep all the default settings. You will use the enzyme lists on the right side to select which enzymes you want the program to use for the digest. In either of the columns (it doesn't matter which one) choose the following enzymes: BamHI, EcorI, HindIII, SmaI, SpeI, XbaI and XhoI. You can select multiple enzymes by keeping your control (Microsoft) or command (Mac) key pressed while clicking on the different enzymes.
Screenshot of the 'Restriction Enzyme Selection' section. 'Select Explicitly' selects which enzymes you want the program to use for the digest. 'Simulate multiple digestion' will allow multiple enzymes to be selected including: BamHI, EcorI, HindIII, SmaI, SpeI, XbaI and XhoI. On this screen, you can also filter selections using a REBASE file.
Figure 5. Screenshot of the "RESTRICTION FRAGMENTS" section where you select your desired enzymes and see the resulting DNA fragments.
- Once you have selected the different enzymes you should notice that different fragments start to show up in the middle section next to the enzyme lists. On the left side you will also see an image that shows your piece of DNA after it has been "cut" up by your selected restriction enzymes (Figure 6). This is called a DNA gel. Each line (called a "band") is a separate, small piece of cut-up DNA. The pieces of DNA are sorted by size from 100 to 10000 base pairs (bp), with bigger pieces at the top and smaller pieces at the bottom. The left lane on the gel (labeled "M") represents a DNA ladder that contains DNA fragments with sizes ranging from 100 to 10000 base pairs. The DNA ladder is used to assess the size of the cut DNA fragments in your DNA sample. The right lane on the DNA gel (labeled "S") represents your sample and shows the different DNA fragments in your cut DNA sample. More detailed information about the DNA bands or fragments, such as which restriction enzymes generated the fragments, is given in the middle section next to the gel image (Figure 5). Below the gel image, click "save the image" to download the image of the DNA gel. You will need this image to compare it to other DNA gels later.
DNA gel
Figure 6. Screenshot of the DNA gel image showing the DNA ladder on the left lane and the different fragments of the cut DNA as individual bands on the right lane.
- In the last section of the tool titled "ANNOTATED SEQUENCE" (Figure 7) you can review your full DNA sequence and see where the different cutting sites for all the different restriction enzymes are located within the sequence.
Screenshot of analysis section with 'Pseudo Gel Map' selected, and a range of 100 to 1000 base pairs.
Figure 7. Screenshot of the "ANNOTATED SEQUENCE" showing the full DNA sequence with all the existing restriction enzymes cutting sites.
- Print the DNA gel image that you downloaded, cut it out, and paste it into your lab notebook for your records. Be sure you label the fingerprint with the name of your DNA (Suspect #1) and that you know the top from the bottom!
- Now you are ready to make a new "Suspect" DNA sequence.
- Repeat steps 1–12 with a new DNA sequence. Just go back to the Random DNA Sequence Generator and start over. This time name your DNA sequence something new (like "Suspect #2" for example).
- Repeat this experiment at least five different times. Each time you will make one new piece of "Suspect" DNA and make a new DNA fingerprint.
- Compare all of your sequences and fingerprints. Look at the pattern of the bands on your different DNA gels. Do they match or are they different? Do unique sequences of DNA result in similar or different DNA fingerprints?
Ask an Expert
Variations
- In this experiment, you are making a new, randomly generated sequence of DNA each time. In reality, our DNA changes very little from person to person. Can small changes in DNA sequences also result in unique DNA fingerprints? To test this, start with one piece of randomly generated DNA and make a fingerprint. Then, instead of making a new DNA sequence from scratch, only change a few nucleotides (letters) of the first sequence. For example, change the middle 10 letters to something new, but leave the rest of the sequence the same. What happens if you make these small changes in different places of the sequence?
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