http://bcs.whfreeman.

com/lehninger5e/

DNADeoxyribonucleicAcid
RNARibonucleicAcid

Discovering the structure of DNA

Structure was discovered in 1953 by James
Watson and Francis Crick

Outline for Replication

A. Initiation
B. Priming
C. Elongation
D. Proofreading and Termination

5 3

Identical

base
sequences

Watson/Crick proposed mechanism of DNA replication

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)

Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:

SSB Proteins
Okazaki Fragments
ATP
1

Polymerase III

2
3

Lagging strand

Helicase
+
Initiator Proteins

3
primase
Polymerase III

RNA Primer
3

base pairs

5
RNA primer replaced by polymerase I
& gap is sealed by ligase

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.

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
HereisanexamplecomparingdATPandddATP:

dATP

ddATP
NH2
N

O
-O

P
O-

P
O-

O
H

O
-O

O-

NH2

OH

P
O-

P
O-

O
O

O-

The3hydroxylhasbeenchangedtoahydrogeninddNTPs,
whichterminatesaDNAchainbecauseaphosphodiesterbond
cannotformatthis3location

MechanismofDNApolymerization
O
O
-O

-O

Base

O-

O
H

O-

DNApolymerase
catalyzed
nucleophilicattack
ofthe3OHona
phosphoanhydride

OBase

O-

O
H

H
O

Base
O
H

H
H

O-

O-

OBase

O-

O-

O
O

P
O-

OBase
O

: OH

H
O

OBase

O
O

H
H
H

-O

O
H

Base

O
Base

O
H

O-

-O

P
O-

OH

OH

O-

H
OH

**Sincethe3OHischangedtoaHinddNTPs,itisunable
toformaphosphodiesterbondbynucleophilicattackonthe
phosphate,anditwillcauseaterminationintheDNAchain

Sequencing Visualization Methods

Two forms of labeling:
Radioactive
Primer labeled (32P or 33P)
dNTP labeled (35S)

Nonradioactive
Primer labeled
ddNTP labeled (big dye terminator)

RadioactivePrimerLabeledSequencing
1.Unknownfragment

6.OnetypeofddNTPperreaction

2.withregionofknownsequence

7.DNApolymerase

3.Complementaryprimer,
5endlabeledwith32Por33P

8.ddNTPincorporation
stopschainsynthesis
Remembereachreactionhasmany
moleculeseachoneincorporating
itsrespectiveddNTPandstopping
atadifferentlength.

4.dNTPs
dATP,dGTP,dCTP,anddTTP

5.Fourseparatereactions
ddATP
5

ddGTP
3

5
5

Reaction1

ddCTP

5
5

Reaction2

ddTTP
3

Reaction3

Reaction4

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.

GelElectrophoresisandReadoutofReactionProducts:
vs.
Radioactive
Sequenceofunknownfragment

ddGTP

ddATP

ddTTP

ddCTP

Longestsynthesizedband=
3endofsynthesizedstrand

Shortestsynthesizedband=5endofsynthesizedstrand

Polymerase Chain
Reaction (PCR) and Its
Applications

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.

What is PCR? :
Why Polymerase?
It is called polymerase because the
only enzyme used in this reaction is
DNA polymerase.

What is PCR? :
Why Chain?
It is called chain because the
products of the first reaction become
substrates of the following one, and
so on.

What is PCR? :
The Reaction Components
1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the sequence
to be amplified.
3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.
4) Thermostable DNA Polymerase - enzyme that
catalyzes the reaction
5) Mg++ ions - cofactor of the enzyme
6) Buffer solution maintains pH and ionic
strength of the reaction solution suitable for the
activity of the enzyme

The Reaction

PCR tube

THERMOCYCLER

Denature (heat to
95oC)

Lower temperature to 56oC

Anneal with primers

Increase temperature to
72oC DNA polymerase +
dNTPs