A Magnetic Primer Designer

Abstract

How do scientists "copy" DNA? They use a process called the Polymerase Chain Reaction, or PCR. The key to making this process work is having a primer that will stick to the piece of DNA you want to copy, called a template. In this experiment you will test how the number of matches and mismatches in a primer will affect its ability to stick, or anneal, to the DNA template during PCR.

Objective

In this experiment you will test how matches and mismatches affect the ability of primers to stick to the DNA that is copied during PCR.

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. Here is an example of a DNA sequence that is 12 base pairs long:

Notice that this piece of DNA has two sequences: one on the top, and one on the bottom. DNA is double stranded, which means that it has two strands. The nucleotides of each of these strands are paired together in a particular way to match the other strand: A pairs with T and C pairs with G. If a nucleotide is paired according to these rules, it is called a match. But if the nucleotide is not paired properly, then it is called a mismatch. Matches and mismatches can affect how the two strands of DNA pair together, and sometimes a mismatch can lead to a mutation.

The information stored in the DNA is coded into sets of nucleotide sequences called genes. Each gene has a set of instructions for making a specific protein. The protein has a certain job to do, called a function. Since different cells in your body have different jobs to do, many of the genes will be turned on in some cells, but not others. For example, some genes code for proteins specific to your blood cells, like hemoglobin. Other genes code for proteins specific to your pancreas, like insulin. Even though different genes are turned on in different cells, your cells and organs all work together in a coordinated way so that your body can function properly.

What if there is something wrong with one of your genes? This can cause problems for your body and how it functions. For example, people who have type I diabetes have problems making insulin. To help people with diabetes, scientists figured out a way to make insulin that diabetics can inject into their body. The insulin is made by a bacteria that has the human gene for insulin.

For scientists to study a gene, they need to be able to isolate it. The simplest way to isolate a gene is to clone the gene into a bacteria, but first you need many, many copies of the gene you want to clone. How do you make copies of DNA? The Polymerase Chain Reaction (PCR) makes copies of a DNA template in four main steps:

• Melt - a high temperature (95^oC) will melt (separate) the two strands of the DNA you want to copy (called the template)
• Anneal - the primer will stick to (anneal to) the template strand
• Extend - the enzyme will copy (replicate) the template DNA strand starting with the primer
• Repeat - the process will be repeated over and over, usually about 30 cycles

PCR is like a copy machine for DNA! (Copyright © The Royal Swedish Academy of Sciences, 2003)

If these first three steps are repeated 30 times, a scientist can make 1 billion copies of a single piece of DNA! That provides the scientist with plenty of DNA material to clone and study. PCR is a very important step in the discovery and manufacturing of genes that become important pharmaceuticals, like the insulin gene.

Notice that if the primer doesn't stick, then the enzyme won't have a place to start copying the template DNA, so designing a good primer is a very important first step for PCR success. In this experiment you will build a model of a primer sticking (annealing) to a DNA template strand using magnets. How important is it for the strength of the primer for the sequence of the primer to match the template strand? You will make matches (where the magnets will stick to each other) and mismatches (where the magnets will repel each other) to model nucleotides in the primer annealing to the DNA template. Will more matches make the primer stick better than a primer with mismatches?

Terms, Concepts and Questions to Start Background Research

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
• Polymerase Chain Reaction (PCR)
• Nucleotide (A, T, C, G)
• Match
• Mismatch
• Anneal

Questions

• How do scientists make copies of DNA?
• What does a primer do, and how does it anneal?
• How will matches or mismatches affect the ability of the primer to anneal to the DNA of interest?

Bibliography

• Check out this site from the Cold Spring Harbor Laboratory for two animations of DNA being amplified and to watch interviews with the inventor of PCR, Kary Mullis:
  DNAi, 2003. "DNA Interactive: Manipulation: Techniques: Amplifying" DNA Interactive (DNAi), Dolan DNA Learning Center, Cold Spring Harbor Laboratory, NY. [accessed March 6, 2007] http://www.dnai.org/b/index.html
• Half of the 1993 Nobel Prize for Chemistry was awarded to Kary Mullis for his invention of the polymerase chain reaction (PCR) method. Try playing this game at NobelPrize.org to learn more about how PCR works:
  Backman, A., 2007. "The PCR Method: A DNA Copying Machine," NobelPrize.org [accessed March 6, 2007] http://nobelprize.org/educational_games/chemistry/pcr/index.html
• Learn what DNA is and what is does from the Genetic Science Learning Center:
  The Tech, 2004. "What is DNA?" The Genetic Science Learning Center, University of Utah, Salt Lake City, UT. [accessed March 6, 2007] http://learn.genetics.utah.edu/units/basics/tour/dna.swf
• Visit this online exhibit at The Tech Museum of Innovation to understand and visualize where the DNA in your body is: Rosa, C. et. al. 2007. "Zooming Into DNA". The Tech Museum of Innovation, San Jose, CA. [accessed March 6, 2007] http://www.thetech.org/genetics/zoomIn/index.html

Materials and Equipment

• Small, flat magnets like the Master Magnetics Ceramic Disc Magnet Value Pack (Model# 07049, 51 Pieces)
  • Available for order online from Amazon:
    http://www.amazon.com/Master-Magnetics-Ceramic-Magnet-Value/dp/B000IYKKV2
  • You can also buy them at a local hardware store like ACE or Home Depot.
• Clear packaging tape, 2 inches wide
• Paint pen in a light color (yellow or white for example)
• Hole punch
• Paper clip
• Dixie cup
• Pennies
• Metric ruler

Experimental Procedure

1. Arrange the magnets in a stack to identify the north and south pole ends of each magnet. Remember that opposites attract, so your magnets will be arranged in an opposing order: N/S, N/S, N/S, etc.
2. Indicate which sides have similar poles by painting the corresponding side of each magnet with a paint pen. When you arrange the magnets in a stack again, the colors should be alternating. (If the magnets you are using already have the poles indicated, then you can skip this step.)
3. The painted side of each magnet will be designated + (plus) and the unpainted side will be - (minus).
4. Next, you will make a simplified model of a DNA strand that you will copy using PCR.
5. Lay out a strip of clear packaging tape (20 cm long), sticky side up, on a table.
6. Place ten magnets along one side of the tape with the painted sides up, spacing the magnets 2 cm apart. The sequence for the DNA strand will be either all plus (+ + + + + + + + + +) OR all minus ( - - - - - - - - - - ) depending upon which side is facing forward. In the example below, let's assume the strand is facing with the negative side towards you (the all minus side).
7. Fold the tape over the magnets, creating a long strip of magnets embedded inside the clear tape. Your model DNA strand should look like this:

8. Now you are ready to make your "primers" which will be shorter versions of your DNA model. Each primer will be 5 magnets in length, but the sequence of the primers will be different.
9. Place a strip of clear packaging tape (10 cm long), sticky side up, on a table.
10. Arrange 5 magnets along one side of the tape, alternating the poles of the magnets in any order you choose. It is important for the positions of the magnets in your primer to be in the same places as the magnets in your DNA strand. Your model primer strand should look like this:

11. Repeat, making more primers with different sequences. It is important to include a positive control primer (+ + + + +) and a negative control primer ( - - - - - ) which can actually be the same primer flipped over!
12. Write the sequences of the primers in a data table, here is an example:

    Primer Name	Primer Sequence	Number of Matches	Number of Mismatches	Number of Pennies
    Positive	+ + + + +	5	0
    Primer 1	+ + + + -	4	1
    Primer 2	+ + + - -
    ...
    ...
    Negative	- - - - -	0	5

13. To test the strength of each primer, you need to add weight to the primer while it is sticking (annealing) to the DNA strand. Then you will increase the weight until the primer falls off. You will do this by attaching a small paper cup to the end of the primer and adding pennies.
14. At one end of the primer strand, use the hole punch to punch a hole near the end of the tape strip.
15. Unfold a paperclip to make a hook, and slip it through the hole of the primer strand and then through the paper cup, so that the cup will dangle from the end of the primer strand. The primer, with hook attached, should look like this:

16. Now you will stick each "primer" to the "DNA" sequence and see if the two strands anneal, or stick together. Count the number of matches and mismatches between your primer and the DNA strand. Write this number in your data table. Here is an example of a primer with one mismatch at the end. See how the magnets repel each other at the end of the strand?

    
    Here is another example of a primer with two mismatches in the middle. See how the magnets repel each other in the center causing a bulge?

17. Hold the DNA strand by one end so that the end of the primer with the paper cup attached will dangle from the other end. Add pennies, one at a time, until the primer falls off. While you are adding pennies to the cup, your experiment will look like this:

18. Write the number of pennies you added in the data table and then test the next primer. Continue testing the primers until you have tested them all. Here are the results for the first two primers tested from the examples above:

    Primer Name	Primer Sequence	Number of Matches	Number of Mismatches	Number of Pennies
    Positive	+ + + + +	5	0	50
    Primer 1	+ + + + -	4	1	33
    Primer 2	+ + + - -
    ...
    ...
    Negative	- - - - -	0	5

    TIP: You can repeat your tests for each primer several times and then calculate an average to get better, more reliable data.

19. Make a line graph of your data, placing the number of pennies on the left side (y-axis) and the number of mismatches on the bottom (x-axis) of the graph.