High Sensitivity Sanger Sequencing for Minor Variant Detection
Introduction to Sanger Sequencing
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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
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Introduction to Sanger Sequencing
Transcript of Introduction to Sanger Sequencing
Introduction to Sanger Sequencing
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=teV62zrm2P0http://www.youtube.com/watch?v=4jtmOZaIvS0&feature=relatedThe 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, well play the sequencing game!
http://wustlysp.pbwiki.com/Genetics+and+Genomics+Teaching+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.
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