|
|
| Goal of lesson - Learners will discover how scientists determine the sequence of a gene. |
Objectives
- The Building Blocks of Life.
- General Concept behind Sequencing.
- Making the Stacks to Copy the Original Sequence.
- Electrophoresis to Separate out the Copied Stacks.
|
| |
| The Building Blocks |
The building block lesson will enable you to learn about how scientists determine the sequence of a gene. DNA contains the important information for almost every living thing. You may recognize DNA as the double helix shown in reference to the subject of genetics.
DNA sequences can be determined through a process known as gene sequencing. This process allows scientists to determine unknown DNA using modified nucleotide bases. This process is based upon the Sanger Method which won its developer a Nobel Prize.
Gene sequencing is also a fundamental component of genomics. Genomics is the science of unraveling the sequence of a large portion of the DNA helix in a given organism. By unraveling the sequence, scientists can identify genes and clusters of genes. Genomics has led to discovery of genes responsible for (1) disease, (2) growth and development, and (3) other biological processes. |
| General Concept |
G = Blue Blocks, C = Green Blocks ,
A = Red Blocks, T = Yellow Blocks |
We use a LEGO® block analogy to explain how genes are sequenced. Each of the different LEGO® blocks in this exercise respectively represent a DNA nucleotide G, A, T, or C as shown on the left. When we examine the genes of an organism, these letters refer to guanine, adenine, thymine, and cytosine, which make up the DNA double helix.
There are two kinds of LEGO® blocks used in our laboratory. LEGO® blocks with their tops and LEGO® blocks without their tops (see directly below). These LEGO® blocks without their tops cannot have other LEGO® blocks attached to them. Thus, the LEGO® blocks without their tops prevent any more blocks from being added to a LEGO® block stack just as the altered (dideoxy) DNA base prevents any more bases from being added to the end of a DNA.
 When we sequence a gene we need to start with a "template" copy of the DNA. This is shown in the large figure to the right labeled "Template." The template DNA is what we will use to determine the sequence of the DNA. In the following lesson we will explain how DNA is sequenced using an analogy where we determine the order of LEGO® blocks in a stack of these blocks. In order to determine the sequence of a gene (or LEGO® blocks) we will use two techniques together.
First, we will make copies of the template. We will make four separate "reactions" to make copies of the template LEGO® stack. Each reaction allows us to determine where each of the four different colored blocks occurs in the stack. In each of these reactions we will have some of the blocks with their tops removed. When one of these blocks, with the top or connector removed, is placed on the stack no more blocks can be added to that stack. Each of the four reactions will have different colored blocks with some of their tops removed: blue, green, red, or yellow. For example, in the first reaction we will have a series of stacks that will stop where the blue blocks occur in the template. In the other three reactions the blocks will stop respectively at the green, red, and yellow blocks in the stack.
After these stacks of blocks are made, we will separate them out based on their size. This separation will be done through a technique known as "electrophoresis" which is analogous to moving the stacks of blocks through bowls of Jell-O® using magnetic charges. This is discussed in more detail in the Electrophoresis section below. Using these combined techniques (LEGO® blocks without there connectors and electrophoresis) we can determine the "sequence" of the colors of the blocks in the LEGO® stack. Both of these techniques are analogous to what happens when a gene is sequenced. The following exercise will outline the details of both of these analogies to explain the basic concepts of gene sequencing. At the end of this lesson you can explore the next level concepts involved in sequencing of genes
|
| Making the Stacks |
|
 Here we can see (figure to the right) that all of the LEGO® blocks are unattached to each other and that all four colors or nucleotide bases are represented in the box.
As part of this lab, LEGO® blocks are selected from the box to build towers. All of the LEGO® blocks will be removed from the box and used to construct several towers which will differ in height.
DNA polymerase is the enzyme that synthesizes DNA from single bases when making a new helix. Some bases are complete, like the normal LEGO® blocks and some are modified, like the flat-topped LEGO® blocks. When DNA polymerase selects a base randomly, it has a chance to choose modified or normal nucleotide bases.
 If the polymerase selects a modified base, then it can't add any other bases to the stack. Thus, construction of the stack, or DNA fragment, ends. It is important to note that DNA polymerase starts again and again continuing until it randomly selects a modified base. By this method, several fragments, each of differing lengths are created.
Each of the LEGO® blocks in this tower are connected to each other. The order of the colored LEGO® blocks tells researchers and scientists important information about the organism that they are studying.
The top LEGO® block in the stack, the one missing its top, represents the end of the stack or sequence. The size or length of this tower is key to learning the genetic code.
The stacks created with LEGO® blocks represent fragments of DNA synthesized by DNA polymerase. These fragments can help researchers to identify the DNA sequence of an organism, such as a plant or humans. |
| Electrophoresis |
|
When all of the LEGO® blocks have been selected from the box and several block stacks constructed, you can look at the different sizes and color combinations of these towers. These stacks represent DNA fragments of different lengths and ending with differing bases.
Scientists use electrophoresis to determine the length of the DNA nucleotide sequences. Electrophoresis is a process commonly used to provide genetic information in many scientific fields, by helping scientists determine the DNA sequence of an organism.
Electrophoresis works by seperating proteins or nucleic acids by size using electric charge. DNA or RNA are typically placed into an agarose gel and an electric gel charge is applied.
The DNA or LEGO® block sequencing fragments are placed into the gel furthest away from the electrical or magnetical source.
Thus, all of the LEGO® block stacks are put into a pan of JELL-O®. A pan of JELL-O® is very similar to the agarose gel used in a laboratory. When studying DNA fragments, all fragments from an experiment are placed in the gel in the same place all at the same time.
We can explain how DNA moves through the gel by using an analogy where negatively charged magnets are attached to the LEGO® block stacks. The blocks are placed in the pan of JELL-O® and then a magnet is placed at the opposite end of the pan with the positive charge of the magnet facing the LEGO® block stacks. The DNA (or LEGO® block stacks) moves towards the positive charge just like two magnets pull each other together when the positive and negative charges face each other.
In laboratories, electric charge is applied to the gel. DNA is composed of molecules that are naturally negatively charged. These molecules are referred to as ions. As positive electrical charge is applied to one end of the gel, the negatively charged DNA moves through the gel.
Once all of the DNA fragments have been placed into the gel and the electric current is applied, researchers can take a picture of the gel to evaluate the results.
 As we can see in the figure to the left, the smallest fragments traveled the farthest through the gel (bottom of the figure) while the longest fragments remained closest to the starting point (top of the figure). This is due to the resistance that the fragment must overcome in order to travel through the gel. The longer fragments experience more resistance than the smaller fragments.
|
| Genomics Animation |
|
To view a short animation that will help you understand how the LEGO® blocks move through the pan of JELL-O®, click here. The concept for this animation is courtesy of Dr. Alan York (video created by Maria Grimes).
We can see that the single person traveled the farthest through the gel, resembling the smallest fragments of DNA or the shortest stacks of LEGO® blocks. Also, we notice that the longest chain of people remained closest to the starting point. This resembles the longest fragments of DNA or the tallest stacks of LEGO® blocks. The difference in each chain's final position is due to the resistance that the chains of people must overcome in order to travel through the crowd. The longer chains experience more resistance than the smaller chains, just as the longer fragments experience more resistance than the shorter fragments. |
| The Building Blocks
|
 Each of the LEGO® block stacks represents fragments of DNA (see figure to the right). All of the top blocks are blue in this example. We know that these top blue blocks are modified nucleotide bases. The black bands, from the picture of the agarose gel, correspond to each fragment that are produced in the "blue block" reaction mix. These stacks define the location of the blue blocks in the template stack. The location of these black bands in the gel picture indicate the size of the LEGO® block stacks (DNA fragments).
Below is the picture of the gel and we can observe that the bands from the gel represent each block in the LEGO® block stack. The LEGO® block stack represents the original strand of DNA. When we look at the "bands" on the below right "gel" it is what we would see if we looked at a DNA sequencing gel.
We know that each of the LEGO® blocks above represents a base pair. We can read the DNA sequence (above right figure) by reading the location of the band in the gel. By the block color the sequence would be as follows: blue (G), green (C), red (A), blue (G), green (C), blue (G), red (A), yellow (T), red (A), blue (G), green (C), and blue (G).
|
The Conclusion |
You have successfully completed the initial LEGO® block lesson. Let's review what you have learned. The sequence of DNA can be determined through a process known as gene sequencing. Gene sequencing, an important part of genomics, has led to countless advances in modern science.
The most noteworthy advance has been the "Human Genome Project." Scientists have successfully identified the entire human genome which includes the sequence of almost all of the human genetic material. The magnitude of this discovery could change how we approach human health.
In agricultural research, genomics has led to advances in genetically modified food products. Genetically modified foods can make the production of food more efficient, nutritious, or even include medicinal properties. The scope of applications for genomics in all areas of science is enormous.
Please proceed to the laboratory exercise. You will now have the opportunity to see how scientists learn the sequence of DNA for yourselves!
If you wish to explore gene sequencing further, please click here.
LEGO® is a trademark of
the LEGO Group
JELL-O® is a trademark of
Kraft Foods, Inc.
|
|
|
Back to Top
|
|
|
> Lesson 3: The Building Blocks of a Gene 
