Download Sequencing

Sequencing
5.
Which reagent don’t you have? DNA polymerase
Phosphodiester
bond
Hydrogen bond
6.
Each cycle of the sequencing reaction has distinct three steps, each performed at a different
temperature:
First Temperature:
96 degrees
Second Temperature: 50 degrees
Third Temperature:
7.
60 degrees
Action: Denature the DNA
Action: Attach primers
Action: Binding of enzymes to the DNA
What causes the DNA to denature in a sequencing reaction?
The heat makes DNA’s molecules vibrate faster than they would at lower temperatures. This
heat-induced movement causes the two strands of DNA to separate.
What type of bonds form between the complementary bases? Hydrogen bonds.
Circle the difference(s) between the structures.
Direction of
DNA synthesis
A deoxynucleotide
triphosphate (dNTP)
A dideoxynucleotide
triphosphate (ddNTP)
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Why do you think a ddNTP would be unable to form a phosphodiester bond?
ddNTPs are missing a hydroxyl (-OH) group on the sugar that plays a major role in the
formation of phosphodiester bonds.
14. Dideoxynucleotide
Color
ddG
yellow/black
ddA
green
ddT
red
ddC
blue
0
1
2
3
4
5
PEAK___________________________________________________________
NORMAL________________________________________________________
blue
0
1
red
black
green
3
4
2
blue
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15. Based upon your sequencing data above, what is the sequence?
CTGAC
16. If your template strand is 10 nucleotides long, how long should your sequence be?
Five nucleotides. The first five letters of the template correspond to the primer.
17. Why is your final sequence different from your original template sequence?
It is the complement of the template sequence.
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