Introduction to Sanger Sequencing
DNA polymerase is an enzyme, which catalyzes the
polymerization of deoxyribonucleotides into
deoxyribonucleic acid (DNA).
There are at least five different polymerases in
prokaryotes and thirteen in eukaryotes! Each has
specific roles. What could some of these roles be?
Below is a prokaryotic Pol III holoenzyme (picture
from neb.com). Label the following parts: Pol III
core, thumb, fingers, active site, processivity
factor (sliding clamp), template DNA, replicate
DNA, deoxyribonucleotides (dNTPs), phosphate,
nitrogen base
Watch the following videos:
http://www.youtube.com/watch?v=teV62zrm2P0
http://www.youtube.com/watch?v=4jtmOZaIvS0&feature=
related
The diagram on
the right
summarizes the
functions of DNA
polymerase (from
wikipedia.com).
What is being
incorporated into
the replicate
strand?
Where does the
energy come from
to produce this
reaction?
How often does
DNA polymerase
incorporate the
wrong nucleotide
(without
proofreading)?
What happens when
there is an error
in extension?
Now with
proofreading, how
often does a
mistake occur?
Here is the normal reaction of a nucleotide being
incorporated into a chain of DNA (www.devbio.com):
By the 1970s, Frederick Sanger used
dideoxynucleotides (ddNTPs) and gel electrophoresis
to actually interpret the sequence of DNA. How are
ddNTPs different from dNTPs
(http://askabiologist.asu.edu)?
Since ddNTPs do not have an OH group,
polymerization stops when a ddNTP is incorporated
into the newly replicated DNA molecule. This is
called chain termination. On the next page is a
summary of how chain termination is used to
sequence DNA.
After we understand the summary, we’ll play the
sequencing game!
http://wustlysp.pbwiki.com/Genetics+and+Genomics+Te
aching+Team
Further Reading
Griffiths, Anthony JF, Jeffrey H. Miller, David T.
Suzuki, Richard C.
Lewontin, and William M. Gelbart. An
Introduction to Genetic Analysis. 7th ed. New
York: WH Freeman and Company, 2002.
Mathews, Christopher K, KE van Holde, and Kevin G.
Ahern.
Biochemistry. 3rd ed. San Francisco: Addison
Wesley Longman, Inc., 2000.
Watson, James D, Tania A. Baker, Stephen P. Bell,
Alexander Gann,
Michael Levine, and Richard Losick. Molecular
Biology of the Gene. 5th ed. San Francisco:
Benjamin Cummings, 2004.