DNA Replication: An Overview

DNA Replication: An Overview
Different models for DNA replication

Semiconservative Replication of DNA as
suggested by Watson and Crick
The Meselson-Stahl Experiment (1958)
The Meselson-Stahl Experiment Supports
Semiconservative Replication
Meselson and Stahl grew a culture of E.coli for many generations in a medium that
contained 15N as the sole nitrogen source. (15NH4Cl).
After many generations, all the E.coli cells had 15N incorporated into the purine and
pyrimidine bases of their DNA.
Meselson and Stahl took a sample of these bacteria and switched the rest of the
bacteria to a medium that contained only 14N (washed them before transferring to
remove the medium containing 15N).
They purified the DNA sample collected from the bacteria just before transfer to the 14N
containing medium.
Meselson and Stahl collected some of the bacteria after each division and extracted
DNA from the bacterial cells (Collected samples of bacteria over the next few cellular
generations).
Under the conditions they used, E.coli replicates its DNA every 20 minutes. They took
samples at an interval of 20 minutes.
After extracting DNA from bacteria they checked for the density of DNA
Outline for Replication
A. Initiation
B. Priming
C. Elongation
D. Proofreading and Termination
Arthur Kornberg
Nobel Prize in 1959
Worked with E. coli. Discovered the mechanisms of

DNA synthesis in 1956.
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. Mg2+ (optimizes DNA polymerase activity)
Polymerization of nucleotide triphosphates
by DNA polymerase – needs free 3’OH
OH
OH
Problem -1
DNA is double helical. Hence the two strands
have to be separated from each other
Problem - 1 : DNA unwinding
DNA is unwound by helicase, stabilized by SSB proteins;
supercoiling is relaxed by Topoisomerase
5’ SSB (single strand binding) Proteins
ADP
Helicase ATP
Helicase 3’
5’
Topoisomerase
3’
Problem - 2
DNA polymerase needs a free 3’-OH of a
pre-existing nucleotide to extend the chain
OH
OH
Problem – 2 : Free 3’ OH group required
Primase synthesizes small RNA primers,
DNA Polymerase III extends them
Okazaki Fragments
1
ATP
Polymerase III 2
Lagging strand 3 Helicase
+
Initiator Proteins
3’
5’
Leading strand
Polymerase III 3’

5’
RNA Primer
primase
3’
Problem -3 : RNA primers need to be removed
RNA primers are replaced with DNA by DNA Polymerase I, which has both
RNaseH and DNA Polymerase activity. The gaps are sealed by Ligase
Okazaki Fragments
1 ATP
Polymerase III 2
Helicase
Lagging strand 3 +
Initiator Proteins
3’
5’
Leading strand RNA primer replaced by

3’ polymerase I (inherent RNaseH)
Polymerase III

5’
RNA Primer Gaps are sealed by ligase
3’
DNA Replication: Summary
• DNA is unwound by initiator protein + helicase
• Single stranded DNA is stabilized by single stranded DNA binding
proteins (SSB)
• Supercoiling is relaxed by Topoisomerase
• Small RNA primers are added by Primase (RNA polymerase)
• DNA Polymerase III extends from 3’ ends of primer sequences
– DNA Polymerase can only extend from pre-existing 3’OH group
– Hence synthesis is always from 5’-3’ direction
– It needs DNA template, dNTPs and Mg2+ ions
• Replication is continuous in leading strand and discontinuous in lagging
strands. Small newly synthesized DNA fragments in lagging strand are
called Okazaki fragments
• RNA primers are replaced with DNA by DNA Polymerase I, which has
both RNaseH and DNA Polymerase activity
• The gaps are sealed by Ligase
Laboratory methods for nucleic
acid analysis
DNA Gel Electrophoresis
DNA Gel Electrophoresis
• Agarose gel is used to analyze a mixture of DNA.
• DNA is negatively charged and hence migrates towards the

positive terminal in the applied electric field gradient.
• Different DNA molecules separate according to mass.
• Smaller molecules migrate faster.
• DNA is visualized in the gel by staining with ethidium bromide,

which fluoresces under UV light.
• UV light at 302 nm or 365 nm wavelengths are used.

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

Nobel Prize in 1993
Polymerase Chain Reaction (PCR)
Why “Polymerase” ?
It is called “polymerase” because the only
enzyme used in this reaction is DNA
polymerase.
Why “Chain” ?
It is called “chain” because the products
of the first reaction become substrates
of the following one, and so on.
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
Reaction Cycle and the Machine
Step-1 Step-2 (x 20-35) Step-3
Temperature
Time
30 cycles = 230 i.e. 1,073,741,824 copies
PCR tube Thermocycler

DNA Sequencing
Frederick Sanger
Nobel Prize in 1980
Sequencing: The process by which you determine the exact order of the
nucleotides in a given region of DNA
Why? To decode the genetic information embedded in DNA
How? Through complementary chain synthesis and early termination
The synthesized chains are visualized by methods using:

– Radioactive labels
– Nonradioactive labels
Dideoxynucleotides (ddNTPs)
OH
dNTP
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
Requirements for Sanger-Coulson
Sequencing
DNA to be sequenced must be in single strand form
The region to be sequenced must be flanked by a stretch

of 3’ 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)
(one for each set)
Mechanism of DNA polymerization
OH
OH
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
Sequencing using denaturing gel-electrophoresis
Template 3’ – A T A T C T G T A C C T A G G T G A G T C A G T A C C -5’
Primer 5’ – T A T A G A C
5’ – T A T A G A C A
Differentially 5’ – T A T A G A C A T G G A
elongated 5’ – T A T A G A C A T G G A T C C A
fragments 5’ – T A T A G A C A T G G A T C C A C T C A
5’ – T A T A G A C A T G G A T C C A C T C A G T C A
To each reaction along with the four regular dNTPs, only one of the
four dideoxynucleotides (ddATP in the above reaction) are added
The ddNTP is added to be approximately 100-fold lower in concentration

than the corresponding dNTP
A series of differentially elongated DNA fragments (hence of different

length) will be produced
Can be visualized by labelling the primers with radioactive phosphate (32P

or 33P)
Sequencing using denaturing gel-electrophoresis
Automated sequencing
Automated sequencing
Each dideoxynucleotide used are linked to a fluorescent
molecule that gives all the fragments terminating in that
nucleotide a particular color.
All four labelled ddNTPs are added to a single tube.
The resulting colored DNA fragments are then separated

by capillary gel electrophoresis
The color associated with each peak is detected using a

laser beam.
The DNA sequence is read by determining the sequence

of colors in the peaks as they pass the detector.
This information is fed directly to a computer, which

determines the sequence.
References
Extra resources
Book:
The Double Helix: A Personal Account of the Discovery of the Structure of

DNA - By James D Watson
Videos:
DNA replication
https://www.youtube.com/watch?v=TNKWgcFPHqw
DNA sequencing:
https://www.youtube.com/watch?v=vK-HlMaitnE

http://video.mit.edu/watch/pcr-polymerase-chain-reaction-10948/
https://www.dnalc.org/view/15475-The-cycles-of-the-polymerase-chain-reacti
on-PCR-3D-animation.html

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DNA Replication: An Overview

Different models for DNA replication

Worked with E. coli. Discovered the mechanisms of

Leading strand RNA primer replaced by

• Agarose gel is used to analyze a mixture of DNA.

• DNA is negatively charged and hence migrates towards the

• Different DNA molecules separate according to mass.

• Smaller molecules migrate faster.

• DNA is visualized in the gel by staining with ethidium bromide,

• UV light at 302 nm or 365 nm wavelengths are used.

It was invented in 1983 by Dr. Kary Mullis

2) Pair of Primers - oligonucleotides that define the sequence to

3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.

4) Thermostable DNA Polymerase - enzyme that catalyzes

5) Mg++ ions - cofactor of the enzyme

6) Buffer solution - maintains pH and ionic strength of the

30 cycles = 230 i.e. 1,073,741,824 copies

PCR tube Thermocycler

Why? To decode the genetic information embedded in DNA

How? Through complementary chain synthesis and early termination

The synthesized chains are visualized by methods using:

The 3’ hydroxyl has been changed to a hydrogen in ddNTP’s, which

DNA to be sequenced must be in single strand form

The region to be sequenced must be flanked by a stretch

Reagents needed are:

Since the 3’ –OH is changed to a –H in ddNTPs, it is unable to form a

The ddNTP is added to be approximately 100-fold lower in concentration

A series of differentially elongated DNA fragments (hence of different

Can be visualized by labelling the primers with radioactive phosphate (32P

All four labelled ddNTPs are added to a single tube.

The resulting colored DNA fragments are then separated

The color associated with each peak is detected using a

The DNA sequence is read by determining the sequence

This information is fed directly to a computer, which

The Double Helix: A Personal Account of the Discovery of the Structure of

Polymerase Chain Reaction (PCR)

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