Transcript Sanger-Coulson Dideoxynucleotide Sequencing Lecture 10/30/00 Kwamina Bentsi-Barnes Deisy Mendoza Jennifer Aoki Best printed in color for clarity.

Sanger-Coulson
Dideoxynucleotide Sequencing
Lecture 10/30/00
Kwamina Bentsi-Barnes
Deisy Mendoza
Jennifer Aoki
Best printed in color for clarity
Topics of Discussion
•
•
•
•
Review of last lecture.
Sequencing.
Requirements for Sanger-Coulson sequencing.
Dideoxynucleotides.
– Mechanism of DNA polymerization.
• Sequencing visualization methods.
–
–
–
–
Radioactive primer labeled sequencing.
Radioactive dNTP labeled sequencing.
Fluorescent primer labeled sequencing.
Fluorescent ddNTP labeled sequencing.
• Gel separation.
• Gel visualization.
– Gel electrophoresis and readout
• Relative template quantities for sequencing.
Review of Last Lecture on
10/25/00
• Southern blot
• cDNA probe
• Good vectors
– pGEM -3zf(+/-)
• Lambda
– Replicate independently
– Fairly small
– cos-sites won’t package, endonucleases recognize those
sites
– cos site is required for package either in vivo or in vitro
• Super Cos
– A lot smaller lambda
– Can replicate individually as a plasmid
Sequencing
• Sequencing is the process by which you determine
the exact order of the nucleotides in a given region
of DNA.
• Dideoxynucleotide sequencing is done through
complementary chain synthesis and early
termination.
• The synthesized chains are visualized by methods
using:
– Radioactive labels.
– Nonradioactive labels.
Requirements for SangerCoulson Sequencing
• DNA to be sequenced must be in single strand
form.
• The region to be sequenced must be 3’ flanked by
known sequence.
• Reagents needed are:
– A primer complementary to the known region to direct
chain synthesis.
– DNA polymerase.
– 4 deoxynucleotide triphosphates (dNTPs).
– 4 dideoxynucleotide triphosphates (ddNTPs).
Dideoxynucleotides
Here is an example comparing dATP and ddATP:
dATP
ddATP
NH2
N
O
-O
P
O-
O
P
O-
N
O
O
H
P
O
N
N
N
O
-O
O
O-
NH2
H
H
OH
H
H
P
O-
H
O
P
O-
N
O
O
O
P
O
O
O-
H
H
H
H
H
H
The 3’ hydroxyl has been changed to a hydrogen in ddNTP’s,
which terminates a DNA chain because a phosphodiester bond
cannot form at this 3’ location
N
N
Mechanism of DNA polymerization
O
O
-O
5’
P
-O
Base
O-
O
H
O-
5’
O
O-
DNA polymerase
catalyzed
nucleophilic attack
of the 3’-OH on a
phospho-anhydride
H
P
OBase
O-
O
H
H
H
H
O
O
H
H
O
H
H
H
O
Base
O
H
H
O
P
H
P
O-
O
H
P
OBase
O-
O
H
H
O
H
H
O
H
P
OBase
O
O
O
H
H
H
: OH
P
O-
O
P
O-
O
P
O
H
H
P
OBase
O
O
O
H
H
:
-O
O
H
H
H
3’
O
O
Base
O
O
Base
O
-O
O
H
O-
H
H
P
O-
O
O
P
OH
H
OH
H
H
O-
H
OH
H
H
** Since the 3’ –OH is changed to a –H in ddNTPs, it is unable
to form a phosphodiester bond by nucleophilic attack on the
phosphate, and it will cause a termination in the DNA chain
H
3’
Sequencing Visualization Methods
• Two forms of labeling:
– Radioactive
• Primer labeled (32P or 33P)
• dNTP labeled (35S)
– Nonradioactive
• Primer labeled
• ddNTP labeled (big dye terminator)
Radioactive Primer Labeled Sequencing
1. Unknown fragment
6. One type of ddNTP per reaction
2. with region of known sequence
7. DNA polymerase
3. Complementary primer,
5’end-labeled with 32P or 33P
8. ddNTP incorporation
stops chain synthesis
Remember each reaction has many
molecules each one incorporating
its respective ddNTP and stopping
at a different length.
4. dNTP’s
dATP, dGTP, dCTP, and dTTP
5. Four separate reactions
ddATP
5’
ddGTP
3’
3’
5’
5’
Reaction 1
-
ddCTP
3’
3’
5’
5’
Reaction 2
ddTTP
3’
3’
5’
Reaction 3
5’
3’
3’
5’
Reaction 4
Radioactive Deoxynucleotide Labeled Sequencing
1. Unknown fragment
6. One type of ddNTP per reaction
2. with region of known sequence
7. DNA Polymerase
3. Complementary primer
8. ddNTP incorporation
- stops chain synthesis
4. dNTP’s
labeled dATP or dCTP
35S
What is different about this method?
(hint: look at the colors)
5. Four separate reactions
ddATP
5’
ddGTP
3’
3’
5’
5’
Reaction 1
ddCTP
3’
3’
5’
5’
Reaction 2
ddTTP
3’
3’
5’
Reaction 3
5’
3’
3’
5’
Reaction 4
Fluorescent Primer Labeled Sequencing
1. Unknown fragment
6. One type of ddNTP per reaction
2. with region of known sequence
7. DNA Polymerase
3. Four separate reactions
8. ddNTP incorporation
- stops chain synthesis
4. Fluorescent labeled primer.
Different fluorescent dye per
reaction
5. dNTP’s
What’s the big advantage here?
(dATP, dGTP, dCTP, and dTTP)
ddATP
5’
ddGTP
3’
3’
5’
5’
Reaction 1
ddCTP
3’
3’
5’
5’
Reaction 2
ddTTP
3’
3’
5’
Reaction 3
5’
3’
3’
5’
Reaction 4
Fluorescent Dideoxynucleotide Labeled Sequencing
1. Here we have one reaction vessel,
with four copies of our Unknown fragment.
Don’t forget that this and the all the previous reaction
vessels have millions of our unknown fragment. Why
do you think we’re only showing 4 representatives?
2. A region of known sequence
3. Complementary primer
4. dNTP’s
5’
5’
5’
3’
3’
5. Fluorescent labeled ddNTP’s. Each
labeled with a different fluorescent dye
5’
ddATP
5’
5’
3’
7. Again ddNTP incorporation
stops chain synthesis
ddGTP
3’
(dATP, dGTP, dCTP, and dTTP)
6. DNA Polymerase
3’
3’
ddCTP
5’
3’
3’
ddTTP
5’
One reaction vessel
Now we run our products on gel
Gel Separation
• The reaction mixtures are separated on a denaturing
polyacrylamide gel.
– Denaturing to prevent the DNA from folding up on
itself while it travels through.
– Polyacrylamide to separate the strands which differ in
length by only one nucleotide.
• Each band corresponds to a sequence of DNA which was
terminated by a particular ddNTP.
• This ddNTP is identified by lane in the radioactive method
and by color in the fluorescent method.
• The lowest band on the gel is the shortest. The shorter the
strand, the earlier in the synthetic reaction the ddNTP was
incorporated.
• The lowest band on the gel is at the 5’ end of our
synthesized strand and is complementary to the 3’ end of
our unknown fragment.
Gel Visualization
• Radioactive method which requires four gel lanes,
one for each reaction vessel.
– Readout is done by hand or with a
densitometric scanner.
• Nonradioactive fluorescence sequencing requires
only one gel lane because each nucleotide has a
distinct color.
– The readout process is done by laser scanner
and recorded by computer.
Gel Electrophoresis and Readout of Reaction Products:
Nonradioactive
vs.
Radioactive
ddATP
ddTTP
ddCTP
Longest synthesized band =
3’ end of synthesized strand
Sequence of unknown fragment
Sequence of unknown fragment
ddGTP
Shortest synthesized band = 5’ end of synthesized strand
Relative Template Quantities needed for Sequencing
Most
Less
Least
•Double stranded
plasmid (must
denature) &
standard
sequencing
reaction
•Single stranded
construct such as
phagemid or
M13 & standard
sequencing
•Either double
stranded or
single stranded
construct types
or fragments &
cycle sequencing
•Each molecule
of template used
only once
•Each molecule
of template used
only once
•Each molecule
is used as
template up to
30 times