science of living system ab (part of unit 2)
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Professor A. BasakDepartment of ChemistryEmail:[email protected]
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http://bcs.whfreeman.com/lehninger5e/
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DNA - Deoxyribonucleic Acid
RNA Ribonucleic Acid
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Discovering the structure of DNA
Structure was discovered in 1953 by James
Watson and Francis Crick
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Outline for Replication
A. InitiationB. PrimingC. ElongationD. Proofreading and Termination
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5 3
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Identicalbase sequences
5
5
3
3 5
5 3
3
Watson/Crick proposed mechanism of DNA replication
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1955: Arthur Kornberg
Worked with E. coli .Discovered the mechanisms of DNA synthesis.
Four components are required:
1. dNTPs: dATP, dTTP, dGTP, dCTP(deoxyribonucleoside 5 -triphosphates)(sugar-base + 3 phosphates)
2. DNA template
3. DNA polymerase ( Kornberg enzyme )
4. Mg 2+ (optimizes DNA polymerase activity)
1959: Arthur Kornberg (Stanford University) & Severo Ochoa (NYU)
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3
Polymerase III
Leading strand
base pairs
5
5
3
3
Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:
Helicase+
Initiator Proteins
ATP
SSB Proteins
RNA Primer
primase
2Polymerase III
Lagging strand
Okazaki Fragments1
RNA primer replaced by polymerase I& gap is sealed by ligase
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Sequencing
Sequencing is the process by which youdetermine the exact order of the nucleotidesin a given region of DNA.
Dideoxynucleotide sequencing is donethrough complementary chain synthesis andearly termination.
The synthesized chains are visualized by
methods using: Radioactive labels. Nonradioactive labels.
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Requirements for Sanger-Coulson Sequencing
DNA to be sequenced must be in single strandform.
The region to be sequenced must be 3 flankedby 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).
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Dideoxynucleotides
dATP ddATP
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
Here is an example comparing dATP and ddATP :
N
NN
N
NH 2
O
HH
HH
HH
OPO
O-
O
POP-O
O
O-
O
O-
N
NN
N
NH 2
O
HOH
HH
HH
OPH
O-
O
POP-O
O
O-
O
O-
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DNA polymerase catalyzed
nucleophilic attack of the 3 -OH on a
phospho-anhydride
** Since the 3 OH is changed to a H in ddNTPs, it is unableto form a phosphodiester bond by nucleophilic attack on thephosphate, and it will cause a termination in the DNA chain
Mechanism of DNA polymerization
: :
O
HO
HH
HH
PO
O-
O-
O
HO
HH
HH
PO O-
Base
Base
O
HOH
HH
HH
OBase
O
P-O O-
O-
5
3
O
HOH
HH
HH
OPO
O-
O
POP-O
O
O-
O
O-
Base
O
HO
HH
HH
PO
O-
O
O-
O
HO
HH
HH
PO O-
Base
Base
O
HO
HH
HH
OBase
PO O-
O
HOH
HH
HH
OBase
O
P-O
O-
5
3
OP-O
O-
O
OHP
O-
O
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Sequencing Visualization Methods
Two forms of labeling: Radioactive
Primer labeled ( 32 P or 33 P)
dNTP labeled ( 35 S) Nonradioactive
Primer labeled
ddNTP labeled (big dye terminator)
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Radioactive Primer Labeled Sequencing
4. dNTPsdATP, dGTP, dCTP, and dTTP
ddGTPddATP ddCTP ddTTP
6. One type of ddNTP per reaction
Remember each reaction has manymolecules each one incorporatingits respective ddNTP and stoppingat a different length.
7. DNA polymerase
3. Complementary primer,5end -labeled with 32P or 33P
5
2. with region of known sequence
3
1. Unknown fragment
5
Reaction 2Reaction 1 Reaction 3 Reaction 4
5. Four separate reactions
5 5
5 3 3 3 5 5
5
8. ddNTP incorporation -stops chain synthesis
3 3 3 3
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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 whichwas 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 reactionthe 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.
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Gel Visualization
Radioactive method which requires four gellanes, one for each reaction vessel. Readout is done by hand or with a
densitometric scanner. Nonradioactive fluorescence sequencing
requires only one gel lane because eachnucleotide has a distinct color.
The readout process is done by laser scanner and recorded by computer.
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Polymerase ChainReaction (PCR) and Its
Applications
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What is PCR?
It was invented in 1983 by Dr. KaryMullis, for which he received the NobelPrize in Chemistry in 1993.
PCR is an exponentially progressingsynthesis of the defined target DNA sequences in vitro .
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What is PCR? :Why Chain?
It is called chain because the
products of the first reaction becomesubstrates of the following one, andso on.
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The Reaction
THERMOCYCLER PCR tube
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Denature (heat to95 oC)
Lower temperature to 56 oCAnneal with primers
Increase temperature to72 oC DNA polymerase +
dNTPs
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