Lecture DNA 3- Primary Structure
and Sanger Sequencing
Reading: Lehninger, Chapter 8 and 9 as
indicated in the text notes
Suggested Problems: Chapter 8, #11 and
12 (as well as problems in the notes)
Gel Electrophoresis
Two common types are agarose and polyacrylamide
gel electrophoresis (PAGE).
Agarose is a cross-linked polysaccharide. We will
learn more about its structure later in this course.
Polyacrylamide is a cross-linked polymer of
acrylamide and N,N-methylenebisacrylamide as
shown below:
When acrylamide is polymerized in water, a gel is
formed.
Gel Electrophoresis
In electrophoresis, charged particles migrate
through a medium in response to an electric field.
Solutions of DNA molecules are loaded on top of a gel
in separate wells.
Since DNA molecules are negatively charged, they will
move through the porous matrix of the gel toward the
positively charged anode (anions move toward the
anode). This is shown in Figure T2.2.
Gel Electrophoresis
In agarose and PAGE, the rate of movement of
molecules (mobility) depends on their size due to
the sieving effect of the gel.
The gel is more porous to small molecules than
large molecules. The shortest DNA molecule will
move through the gel more rapidly and can be
found farthest down the gel.
The DNA fragment can be isolated from a slice of
the gel.
What we have learned so far
The information for biological structure and
function is encoded in the nucleotide base
sequence of the DNA molecules in cells. (Primary
Structure)
Naturally occurring DNA molecules are very large.
About 4 million bases are found in each strand of E.
coli DNA.
To determine the sequence of large DNA
molecules, two sample preparation techniques
were necessary:
DNA for Sequencing
1)-Fragment the large DNA molecule into manageable,
defined pieces
Restriction endonucleases (such as EcoR1)
accomplished this step.
2)-Obtain a significant amount of each fragment to
work with.
Molecular cloning (a recombinant DNA technique)
accomplished this step.
Also, defined pieces of the large DNA molecule can be
isolated by the biochemical technique of gel
electrophoresis (separates molecules by molecular
size)
Determining the Sequences of Long
DNA Strands
Two sequencing techniques (1977) were
developed which depend on nucleotide
chemistry, DNA metabolism and electrophoretic
methods:
1)-Sanger - more widespread use, technically
easier
2)-Maxam-Gilbert - uses two basic reactions to
cleave a polynucleotide chain wherever a
particular kind of residue (A, T, G or C) is found.
We will focus our study on the Sanger Method.
Sanger Method (Dideoxy Method) Reading
material-Lehninger, 5th ed., Chapter 8.3 (pp. 292-294)
The Sanger method uses synthetic
2,3-dideoxynucleoside triphosphates and DNA
polymerase (enzyme involved in DNA replication)
to sequence DNA.
The structure of 2,3-dideoxynucleoside
triphosphate is shown in the handout for this
lecture as Figure 3a.
Note in this structure that both the 3 and the 2
carbon atoms of ribose do not have an OH group.
To understand the Sanger Method, we must
first know how DNA is synthesized.
Figure 1. Mechanism of DNA Synthesis by DNA
Polymerases: Chain Elongation
When DNA is synthesized, one strand acts as a
template for the synthesis of the complementary
strand.
An enzyme called DNA polymerase catalyzes the
sequential addition of nucleotides to the 3 end of the
growing strand.
This process requires:
1) a single unpaired strand to act as a template and
2) a primer (short oligonucleotide) strand to provide a
free hydroxyl group at the 3' end, to which a new
nucleotide unit is added.
Chain Elongation
Each incoming nucleotide which is added to the primer
strand is selected by base pairing to the appropriate
nucleotide in the template strand.
For example, A is added if T is in the template, and G is
added if C is in the template (complementary base
pairing).
In cells, the 3'-hydroxyl group of the primer reacts with
the incoming deoxynucleoside triphosphate (dNTP) to
form a new phosphodiester bond.
The fundamental reaction of DNA synthesis by DNA
polymerases is a phosphoryl group transfer.
Chain Elongation
The nucleophile is the 3'-hydroxyl group of the nucleotide at
the 3' end of the growing strand.
Nucleophilic attack occurs at the phosphorous of the
incoming deoxynucleoside 5'-triphosphate.
Inorganic pyrophosphate is released in the reaction as shown:
(dNMP) n + dNTP (dNMP) n+1 + Pp i
DNA
Lengthened DNA
where dNMP and dNTP are deoxynucleoside
5'-monophosphate and 5'-triphosphate, respectively.
Chain Elongation
Hydrolysis of the pyrophosphate released
provides some of the thermodynamic driving
force for the reaction.
The reaction product has a new free 3' hydroxyl,
which allows the addition of another nucleotide.
Fig. 2 shows the mechanism of chain elongation
using complete chemical structures.
Dideoxynucleoside triphosphate (ddNTP)
analogs
The Sanger procedure uses dideoxynucleoside
triphosphate (ddNTP) analogs to interrupt
DNA synthesis.
The structure of ddNTP is shown in Figure 3a.
For ddNTP, Note that both 3' the and the 2' carbon
atoms of ribose do not have an OH group.
When a ddNTP is inserted in place of a dNTP strand,
strand elongation is halted after the analog is added to
the primer strand. Why???
Because ddNTP lacks the 3'-hydroxyl group needed for
the next step.
Fig T20.1 Cloning into M13 and
Sequencing by the Sanger method
CLONING
As noted earlier, a single strand of DNA is
needed for sequencing.
The top half of Fig T20.1 shows how a single
strand of DNA containing the segment of
unknown sequence is obtained from a
double-stranded recombinant DNA
propagated in bacteria infected with a phage.
Sanger Method
SEQUENCING
The single stranded DNA is mixed with an
oligonucleotide complementary to the known
sequence of the plasmid, which is directly next to
the unknown segment.
The oligonucleotide binds to the known sequence
through base pairing and acts as a primer (with
its free 3 OH group) for the synthesis of a new
strand of DNA.
Sanger Method (contd)
Next, four reaction solutions are prepared which
contain the following:
the single strand of DNA and bound oligonucleotide
primer
a mixture of 2'-deoxyribonucleoside triphosphates
an P labeled 2'-deoxyribonucleoside triphosphate
DNA polymerase.
32
The radioisotope of phosphorous is P. Incorporating
this isotopically labeled nucleotide into the synthesized
DNA fragment allows one to determine the position of
the fragment on a gel by autoradiography.
32
Sanger Method (contd)
One of the four 2',3'-dideoxynucleoside triphosphates
is mixed into each tube, at a concentration 100 times
lower than the dNTP concentration.
When a 2',3'dideoxynucleotide is added to the chain,
chain growth is terminated because there is no 3'OH
group for a subsequent nucleotide to react with.
As a result, chain fragments of different length are
randomly synthesized. The reaction producing each set
of labeled fragments is base-specific.
Sanger Method (contd)
In the A reaction solution (contains ddA), the
synthesized fragments all end in A.
Similarly, the G, C and T reaction solutions will produce
new strand fragments which end in G, C and T,
respectively.
Thus, the lengths of the fragments correspond to
positions in the DNA sequence where a certain base
occurs (See the bottom half of Figure T20.1 for
fragments generated).
The length of each synthesized fragment is a
measure of the number of nucleotides from the
primer to the dideoxynucleotide.
Gel Electrophoresis
The four reaction solutions are added to the gel
(one solution in each well) and subjected to
electrophoresis (PAGE or agarose).
Gel Electrophoresis separates the synthesized
fragments by length.
Exposure of photographic film to the gel allows
an identification of bands. Because the fragments
are radio labeled at their 5' ends with P, only the
fragment to the 5' side of the break is visualized.
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Electrophoresis Results- Sanger
Method
A ladder of bands is produced. The gel is read from the
bottom to the top.
Because shorter DNA fragments migrate faster, the
fragments near the bottom of the gel represent the
nucleotide positions closest to the primer (the 5' end).
Thus, reading the gel from the bottom to the top
corresponds to the sequence from the 5 3 end of
the non-template strand (the primer strand).
The sequence of the template strand is the
complement of the sequence read from the gel.
Also note that the 3' end of the non-template strand
corresponds to the 5' end of the template strand.
Sanger Method (contd)
Typically, 250 bases in the sequence can be
determined by the dideoxy method.
Theres a good example in Lehninger, p.
293, DNA Sequencing by the Sanger
Method.
Lets try it together in class. Refer to Figure 4 in
the handout for all of the starting information.
There is also a homework problem at the end of
your packet.
Automating DNA Sequencing
DNA sequencing is readily automated.
In a variation of Sangers method, the
dideoxynucleotides used for each reaction are labeled
with a different colored fluorescent tag.
This technology allows 1000s of nucleotides to be
determined in a few hours.
Figure 8-34 Strategy for Automating DNA Sequencing
Reactions in Lehninger, p. 294 provides a good color
presentation of this process.
In the Human Genome Project, researchers have
sequenced all 3.2 billion base pairs of the DNA in a
human cell.
Summary
The Sanger method requires the enzymatic synthesis of a
DNA strand complementary to the strand being analyzed,
using a radioactively labeled primer and
dideoxynucleotides.
The general principle is to reduce the DNA to four sets of
labeled fragments.
The reaction producing each set is base-specific. The
lengths of the fragments correspond to positions in the
DNA sequence where a certain base occurs.
Electrophoresis of the four sets of fragments produce a
ladder of bands from which the sequence can be read
directly.
The DNA sequencing reactions can be automated, enabling
larger fragments to be sequenced in a few hours.
Topic for Next Lecture
DNA Fingerprinting- Lehninger, 5th edition,
chapter 9, pp. 317-324 (most of section 9.2)