1. Di-deoxynucleotide Chain Termination
DNA Sequencing
Di-deoxynucleotide Chain Termination

2. ...ponder...
What does it mean to “sequence” DNA?
What are some applications of DNA sequencing?

3. Step 1
The DNA to be sequenced is prepared as a single strand by applying heat.

4. Step 2: This template DNA is supplied with:
a mixture of all four normal (deoxy) nucleotides
DNA polymerase
Primer
dATP
dGTP
dCTP
dTTP

5. Step 2:
The reaction mix is added to separate tubes with dideoxynucleotides
ddATP
ddGTP
ddCTP
ddTTP

6. Carbon 2 AND 3 are “deoxy”
Hence “di-deoxy…”
DNA polymerase 3 can NOT add another nucleotide to this one
Carbon 2 is “deoxy”
This is a “normal” DNA nucleotide
DNA polymerase 3 can add another nucleotide to this one

7. Step 3:
Replication proceeds normally until, by chance, DNA polymerase inserts a dideoxy nucleotide instead of the normal deoxynucleotide .

8. Step 3:
ADD COLOR TO YOUR NOTES!!!

9. Step 4:
ADD COLOR TO YOUR NOTES!!!
The daughter fragments are separated in electrophoresis from longest to shortest.
A difference of one nucleotide is enough to separate that strand from the next shorter and next longer strand.

10. Step 4 cont.:
Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser at the end of the electrophoresis chamber
An automatic scanner provides a electropherogram of the sequence.

11. Helpful links Sanger Method reading
DNA Sequencing reading with animation
DNA Sequencing animation #1
DNA Sequencing animation #2 with quiz