In Search of Satellite DNA in Callosobruchus maculatus
Student Handout
Objectives
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Distinguish between heterochromatin and euchromatin in terms of structure and function
Illustrate the process by which restriction enzymes cleave DNA
Describe the process of gel electrophoresis
Analyze banding patterns from DNA digests in an agarose gel
Predict banding pattern in a gel for DNA digested with restriction enzymes
Introduction
What is Satellite DNA?
You are aware that each person’s genetic code is unique. Excluding identical twins,
every single human being has a unique combination of alleles, which is determined by the base
pair sequence in the genome. Because the base pair sequence is different between individuals,
we can use DNA to answer important questions, such as during criminal investigations or
understanding familial relationships. How does this information apply to other species? Why
would it be useful to understand genetic differences between beetle species?
Scientists use a technique called DNA fingerprinting to compare DNA from multiple
individuals (Reece et.al. 2011). Using restriction enzymes, scientists isolate very small, repeated
DNA sequences called short tandem repeats, or STRs. STRs are found in non-coding regions of
the genome, and the quantity of repeats varies between individuals. For example, consider the
STR sequence ATC. In one person, that section of DNA may repeat twelve times, producing a
DNA fragment that is 36 base pairs long; however, in another individual, that sequence may
repeat twenty times, producing a DNA fragment that is sixty base pairs long. When these
fragments are amplified using polymerase chain reaction (PCR), they can be visualized via gel
electrophoresis. Comparing banding patterns based on fragment size would allow you to identify
differences in DNA sequences between individuals.
The challenge of using STRs is that it requires PCR to visualize the differences in
sequence length. Visualizing differences in STR length without PCR is nearly impossible
because the amounts of DNA are so minuscule that bands would be nearly invisible. Short,
“light” DNA fragments do not appear on gels as prominently as longer, “heavier” DNA
fragments. Amplifying the quantity of DNA fragments using PCR is what makes STR bands
visible on the gel. PCR technology is not available to us, and therefore, not possible in this lab
setting. Nevertheless, this does not preclude us from assessing differences in DNA sequences.
While we cannot use STRs, there are other repeated sections of DNA that tend to be
much longer, called satellite DNA. When satellite DNA is isolated using restriction enzymes,
the resulting DNA fragments are sufficiently large to be visualized without using PCR. Satellite
DNA has been reported in many species of beetles, such as Tenebrio molitor (Pons et. al. 2004).
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However, it is not clear at present if the bean beetle, Callosobruchus maculatus, has repetitive
sequences. Satellite DNA comprises of a collection of non-coding tandemly repeating DNA.
This DNA is usually restricted to the centromere region and other heterochromatic structures.
These repeating sequences may be as small as a few dozen to as large as a few thousand base
pairs. Reports show that insect satellite DNA consists of two size classes, 140-190bp and 300400bp (Palomeque and Lorite 2008). While satellite DNA is not used to differentiate between
individuals, it is often used as a taxonomic identification tool since different, though closely
related species, exhibit differing satellite DNA (Ugarković and Plohl 2002).
In this activity, you will apply molecular biology techniques to perform authentic
research and answer these real world questions or any other of your creation:
1. Is satellite DNA present in the bean beetle genome?
2. If present, is there a difference between satellite DNA of different populations? Between
sexes of bean beetles? Between bean beetles and other beetle species?
Materials
At least 50 bean beetles will be used for isolation of genomic DNA. Bean beetles will be crushed
and tissue isolated using solutions and following the protocol provided by the purification kit for
genomic DNA from Promega (kit A1120). Once genomic DNA has been isolated, you may
choose from a battery of restriction enzymes to perform your digest. Restrictions digests will
then be separated by agarose gel electrophoresis and analyzed.
Experimental Design
Preparation & Observations
Before doing the experiments below, take 15 minutes to familiarize yourself with bean beetles.
Practice moving the beetles from one container to another using either your fingers or blunt
tweezers. Take care not to squeeze too hard as you will squish the beetle. Also, use this time to
sex your beetles using the information from the bean beetle handbook. Put the females in one
container, and the males in another if you will be exploring sex differences.
Day #1 – Genomic DNA Purification
On day 1, you will purify genomic DNA. In part 2, you will decide whether to digest the DNA or
not and run your samples on a gel.
Null Hypothesis:
Alternative Hypothesis:
Prediction(s):
Experimental Protocol
It might be more convenient to place the beetles for at least 1 hr before handling them in the
freezer to prevent them from escaping. Select at least 50 beetles per group of students. That
number of beetles should yield enough DNA for 4-6 samples. Crush the beetles in a 1.5 ml
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microfuge tube using a glass rod. Use a toothpick to get to the insects in the tip of the tube. Using
Promega kit A1120 (Purification Kit for Genomic DNA), isolate the DNA. Rehydrate the DNA
in 100 ul of DNA Rehydration solution for 1 hour at 65oC or overnight at room temperature.
Store at -20oC until ready to load or digest. Make sure to label your tube properly so you can find
it next lab period.
Day #2 – Searching for Satellite DNA with Restriction Enzymes
The problem with attempting to visualize the entire genome in a gel is that chromosomes are so
large that they barely enter the gel. Most of the DNA remains close to the loading well.
Restriction enzymes will help you solve this problem by cutting the DNA at specific locations,
hopefully producing unique DNA fragments banding pattern. Of course, by cutting the DNA
you are no longer examining the entire genome, but a very specific section, sections that may
qualify as satellite DNA.
Null Hypothesis:
Alternative Hypothesis:
Prediction(s:)
Experimental Protocol
Use 10-16 ul of rehydrated, thawed, DNA. If appropriate, digest with the enzyme of your choice.
Make sure you add the appropriate buffer for your restriction digestion. After the specified time,
add loading dye and load the digested sample onto a 1% agarose minigel. Remember to include a
control consisting of undigested genomic DNA and a DNA size ladder. Run for 30 min at 100 V.
At this time, your professor will treat your gel with ethidium bromide and will photograph it.
Analyze the photograph of your gel with your lab mates and compare it with published
restriction digest gels (Palomeque and Lorite, 2008; Pons, et. al. 2004).
Results
Draw the image of your gel below, be sure to include a MW ladder in your diagram. Describe
the banding patterns between your samples. How are they different? How are they similar?
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Discussion
Were you able to visualize differences in DNA using this protocol? Were there similarities
between individuals of the same population? Could you use this information to help you
categorize an unknown individual as belonging to one population or the other? Did you reject or
fail to reject the null hypothesis? Explain your answers.
Literature Cited
Palomeque, T. and Lorite, P. 2008. Satellite DNA in insects: a review, Heredity 100, 564–573.
(downloadable from http://www.nature.com/hdy/journal/v100/n6/pdf/hdy200824a.pdf)
Pons, J., Bruvo, B., Petitpierre, E., Plohl, M., Ugarkovic, D. and Juan, C. 2004. Complex
structural features of satellite DNA sequences in the genus Pimelia (Coleoptera:
Tenebrionidae): random differential amplification from a common 'satellite DNA
library’, Heredity 92, 418–427. (downloadable from
http://www.nature.com/hdy/journal/v92/n5/pdf/6800436a.pdf)
Reece, J.B., Urry, L.A., Cain, M.L., Wasserman, S.A., Minorsky, P.V. and Jackson, R.B. 2010.
Campbell Biology (9th ed.). Benjamin Cummings, San Francisco, CA.
Ugarković, D. and Plohl, M. 2002. Variation in satellite DNA profiles--causes and effects.
EMBO J. 21(22): 5955-5959. (downloadable from
http://www.ncbi.nlm.nih.gov/pubmed/12426367)
This experiment was written by Beatriz Gonzalez and Elizabeth Deimeke, 2014
(www.beanbeetles.org).
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