Di-deoxynucleotide Chain Termination DNA Sequencing Di-deoxynucleotide Chain Termination

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

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

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

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

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

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

Step 3: ADD COLOR TO YOUR NOTES!!!

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.

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.

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