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Discovering the structure of DNA Structure was discovered in 1953 by James Watson and Francis Crick
5 3
5 3
1955: Arthur Kornberg Worked with E. coli. Discovered the mechanisms of DNA synthesis. Four components are required:
1.
2. 3. 4.
SSB Proteins
primase
Polymerase III
base pairs
5
RNA primer replaced by polymerase I & gap is sealed by ligase
RNA Primer 3
Leading strand
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.
Dideoxynucleotides
Here is an example comparing dATP and ddATP:
dATP
NH 2 N
ddATP
NH 2 N
O -O P OO
O P OH
O P OH OH H O H
N
-O
O P OO
O P OO
O P OH H H O H
O H H
O H H
The 3 hydroxyl has been changed to a hydrogen in ddNTPs, which terminates a DNA chain because a phosphodiester bond cannot form at this 3 location
5
O H
Base
-O
P O-
5
O H H H
Base
P O-
: OH :
3
O
O H H OH H Base O H H
O -O P OO
O P OO
O P O-
O O P OOH
-O
P O-
** 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
Nonradioactive
Primer labeled ddNTP labeled (big dye terminator)
6. One type of ddNTP per reaction 7. DNA polymerase 8. ddNTP incorporation stops chain synthesis -
Remember each reaction has many molecules each one incorporating its respective ddNTP and stopping at a different length.
ddCTP
5 5 3 3 5 5 3
ddGTP
3
ddTTP
3 5
Reaction 1
Reaction 2
Reaction 3
Reaction 4
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.
ddCTP
What is PCR?
PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro. It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993.
The Reaction
PCR tube
THERMOCYCLER