Name __________________________________ Period _________

Ms. Foglia Date ______________________

AP LAB 23: SANGER SEQUENCING

It is a bit of a conceptual leap from the discovery of the structure of DNA to the sequencing of
the human genome, but one leads directly to the other. The double-helix model of DNA led to an
understanding of how the DNA is duplicated as cells grow and divide. This process of DNA
replication was then harnessed as a tool for the Sanger method of determining the sequence of
a piece of DNA.
Modern DNA sequencing technology is based on the method of controlled interruption of DNA
replication developed by Fred Sanger in 1977, for which he was awarded his second Nobel
Prize in 1980 shared with Walter Gilbert and Paul Berg (he won his first unshared Nobel in 1958
for his work on the structure of proteins, especially that of insulin).
Sanger combined the natural DNA replication
machinery of bacterial cells (with a bit of
recombinant DNA technology and some
clever biochemistry) to create a system in
which a cloned fragment of DNA is copied,
but some of the copies are halted at each
base pair along the sequence.
Natural DNA replication uses the DNA
polymerase enzyme which copies a template
DNA sequence (one half of the DNA double
helix) and creates a new DNA polymer,
complementary to the template, by joining
nucleotides into a growing DNA chain.
Remember that the replication reaction also
requires a primer—a short piece of DNA that
is complementary to the template—to which Figure 1. DNA replication: DNA polymerase
the polymerase can affix the first added base uses a primer and free nucleotides to
(see Figure 1). synthesize a complementary strand of DNA
from the original template strand. Sanger
The Sanger Method sequencing builds on this natural process.

The Sanger sequencing method builds on this natural copying process. This laboratory
technique makes use of specially modified nucleotides — dideoxynucleotides. DNA is called
deoxyribonucleic acid because the ribose sugar part of the molecule is lacking an oxygen atom
found in normal ribose. Dideoxy bases lack a second oxygen atom that is required to extend the
growing DNA chain. This means that when a dideoxy base is incorporated into a DNA molecule,
the chain stops or terminates.
The reactions are set up so that there is a mix of “normal” bases (G, A, T, C) and dideoxy bases
(ddG, ddA, ddT, ddC) as well as the other components necessary for replication: primers and
DNA polymerase enzyme. In this way, the reaction is set up so that it doesn't work all the time
— we don't want a perfect, complete copy of the DNA. At any position, either a normal base will
be added, so the chain can continue to grow, or a dideoxy base will be added, so the chain
terminates. After many cycles of copying, all the possible chain-termination molecules are
produced: the reaction has stopped chains at every base (see Figure 2).

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In the original technique developed by

Sanger, the fragments are separated by
length using polyacrylamide gel
electrophoresis, with one gel lane for
each of the four different
dideoxynucleotide reactions: a G lane,
an A lane, a T lane, a C lane (see Figure
3 and Figure 5a). The synthesized DNA
segments are sorted by size: short ones
travel further than long ones. An
autoradiogram is made of the gel, and
the DNA sequence is visualized and
interpreted.
DNA fragments of a specific length form
a distinct band on the gel, so there is
Figure 2. Sanger sequencing using dideoxy-
one band for each base in the template
nucleotides to terminate growing DNA chains.
sequence. Then the gel, which contains
the radioactively labeled DNA fragments, is placed on top of a sheet of X-ray film so that the
radioactive bands of DNA can expose it (see Figure 4). Finally, the sequence is manually read
off of the X-ray film from the positions of the bands and typed into a computer.
The value of determining DNA sequences was immediately obvious to many biologists, but the
laboratory techniques of the Sanger method, while being ingenious, are both laborious and
technically demanding. DNA sequencing became a rite of passage for many molecular biology
graduate students in the 1980s and early 1990s. Initially, some kits were developed to simplify
and standardize the biochemistry. These kits eventually included superior types of polymerase
enzymes, and minor improvements were made in the polyacrylamide gel apparatus; however,
the essential technique remained unchanged for about 15 years.

Automated DNA Sequencing

The first major innovation to improve
DNA sequencing was Leroy Hood’s
development of fluorescently labeled
nucleotides in 1985 to replace the
standard radioactive labels. Most
commonly, each dideoxy base is given a
different “colored” tag: we can imagine
that A is green, G is yellow, C is blue,
and T is red. Now, the fluorescent labels
could be measured directly in the
polyacrylamide gel as DNA fragments
passed by a laser detector, thus
eliminating both the radioactivity and the
X-ray film. In addition, using the four
differently colored fluorescent labels
meant that the four sets of fragments Figure 3. A sequencing gel showing bands in four
could be run in a single lane of a gel lanes, representing the DNA fragments
polyacrylamide gel and the identity of the produced by the four different dideoxy sequencing.
correct base could be determined

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automatically by the color of the end base on each fragment (see Figure 5a). Hood also directly
connected the fluorescent detector to a computer so that the fluorescent signal was
automatically collected and converted to a DNA sequence (see Figure 5b). Together with Lloyd
Smith, Michael Hunkapiller, and Tim Hunkapiller, Hood founded Applied Biosystems, Inc. (ABI),
which manufactures a commercial version of this automated fluorescent sequencer, which
became available in 1986.
Since 1986, ABI (in cooperation with the PerkinElmer Corp.) has consistently improved their
machines and has dominated the commercial marketplace for automated sequencers.
Essentially all of the Human Genome Project and absolutely all of Celera Genomics’
sequencing was done on ABI machines. However, ABI machines still have many of the
limitations of the original Sanger method. They still rely on DNA polymerase to copy a template
DNA sequence and on polyacrylamide gel electrophoresis to separate the fragments.

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Figure 4. An autoradiogram (X-ray film) of a DNA polyacrylamide sequencing gel. Each

sequence requires four lanes, one for each base.

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a.

(i) (ii)
b.

Figure 5. ABI fluorescent sequencers allow all four bases to be sequenced in a single gel lane
and includes automated data collection directly into a computer.

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Ms. Foglia Date ______________________

AP LAB 25: SANGER SEQUENCING SIMULATION

Objective:
This lab will simulate the Sanger method of sequencing DNA, both visually and kinesthetically; it
uses colored pop-beads, which represent nucleotides and dideoxynucleotides. The
dideoxynucleotides have their "stubs" cut off to simulate the inability of these molecules to
extend the DNA chain beyond them.

Materials:
• A segment of DNA
• Reaction containers with pop-beads representing the 4 different DNA bases:
• green = adenine
• blue = cytosine
• yellow = guanine
• red = thymine
note some of the pop-beads have their ends cut off to represent the
radioactive dideoxynucleotides
• Masking tape
The masking tape is used to tape off a table top in the outline of an electrophoresis chamber
with four wells, labeled A, C, G, and T respectively, to run your reaction mixtures

Teacher Guide:

1. Divide the class into 4 groups.

2. The following reaction container mixtures need to be prepared by the instructor. Each group
of students will only receive one reaction container mixture.
Group
A C G T
Color
Adenine (green) 50, 25 dideoxy 75 75 75
Cytosine (blue) 75 50, 25 dideoxy 75 75
Guanine (yellow) 75 75 50, 25 dideoxy 75
Thymine (red) 75 75 75 50, 25 dideoxy

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Procedure

1. Below is the section of DNA that you will be sequencing. Obviously if this were a true
sequencing situation, you would not know this sequence. You would be using this procedure
to determine the sequence of this unknown segment of DNA. You get to see this sequence
now, because, in this part of the lab, you will be playing the role of DNA polymerase
reading the DNA template.

Figure 1.
single strand
of DNA 3’ 5’ DNA template
C T G A C T T C G A C A
5’ 3’
new sequence primer

2. Fill in the open half of the DNA strand in Figure 1 above with the appropriate nucleotide
letter. Check that these are correct, as these will form the single-stranded nucleotide chains
in your DNA sequencing.

3. Obtain a "reaction mixture" container of nucleotide & dideoxynucleotide pop-beads. There

are four different bags. Each lab group will prepare sequences from only one "reaction
mixture" container. Class results will be combined in the "giant electrophoresis chamber,"
which is taped on one of the lab tables.

4. Start building nucleotide chains as complementary strands to the DNA

sequence in Figure 1. The “nub” on the pop-bead is the 3’ end of the 5’ 3’
nucleotide. Remember, DNA can only be built by adding bases to the
3’ end of a growing DNA strand. Select nucleotides out of your "reaction mixture" container
without looking at whether you have selected a nucleotide or a dideoxynucleotide pop-bead.
Remember, you are DNA polymerase so you must always construct your sequences from 5’
to 3’. Build each strand until you link a dideoxynucleotide pop-bead to the chain. This always
terminates the sequence. (You will quickly see it is physically impossible to add anymore
nucleotides after a dideoxynucleotide has been bonded to the chain.) You will end up with a
variety of DNA sequences of different lengths. Continue until you have 10-12 DNA
segments, or until you run out of nucleotides.

5. Take your DNA segments to the "giant electrophoresis chamber," at the specially marked
lab bench. Place your sample in the proper "well" of the chamber. For example, the group
which works with the reaction mixture containing dideoxyadenine will put its segments into
"well A." Turn on the current (i.e., use your hands and mind!) and move your DNA segments
their proper distance along the "sequencing gel". Remember: Smaller segments migrate
farther than larger segments.

6. When all groups have run their reaction mixture through the "sequencing gel," sketch the
DNA sequence bands in the chamber illustration on the next page. Fill in the proper bases
(letters) on the blanks to the right of the illustration These bases signify the last base — the

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dideoxynucleotide base — that terminated the strand. Since smaller segments of DNA
migrate farther on the gel, the sequence is then read from bottom to top.

DNA Sequencing Gel

A C G T
–

Questions
Complete the following questions by typing your answers on a separate sheet. You may hand
in the document as a print-out or you may send it to me as an attachment to an e-mail.

1. What figurative role did people play in the sequencing technique?

2. What function does the gel play in the sequencing process?

3. Why was the use of colored fluorescent tagging a significant advancement over the use of
radioactively tagged nucleotides?

4. Discuss 3 examples in which a person might want to make use of DNA sequencing
technology.

5. Briefly explain how many advancements in basic biological are enabled only by
advancements in technology.

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