Replication Introduction , DNA replicating Models , Meselson and Stahl Experiments , Circuler Model of DNA replication , Replication in Prokaryotes , Replication In Eukaryotes , Comparison Between Prokaryotes and Eukaryotes Replicaton and PCR (Polymerease Chain Reaction)

2. 1:REPLICATION DEFINITION.
2:REPLICATION INTRODUCTION.
3:REPLICATION HISTORY.

9.  DNA contains all information's for human .
 Before any cell division, a cell must duplicate all its
DNA.
 Replication occurs during S phase of cell cycle
(synthesis phase of interphase).
Mitosis
-prophase
-metaphase
-anaphase
-telophase
G1 G2
S
phaseDNA replication takes
place in the S phase.

10.  Replication means making a replica ( an exact or
identical copy)
 DNA replication is the process of making an exact
copy of DNA by using one of the parent DNA strands
as template.
 DNA functions to :
◦ Store genetic information & transferring it to daughter cells
during mitosis by replication &
◦ Transfer of genetic information from DNA to RNA to be
expressed as proteins

11.  DNA is a polymer of deoxyribonucleotides .
 Deoxyribonucleotides are dAMP, dGMP, dTMP & dCMP.
 A nucleotide is a building block of DNA and is composed of
:a nitrogenous base, a pentose sugar and a phosphoric acid.
 Nitrogenous bases are : Adenine , Guanine, Thymine &
Cytosine

22. MoDeLs oF
DNA
REPLICATION

39. Prokaryotic cells are quite
simple in structure. They
have no nucleus, no
organelles and a small
amount of DNA in the form
of a single, circular
chromosome.
Eukaryotic cells on the
other hand, have a nucleus,
multiple organelles and
more DNA arranged in
multiple, linear
chromosomes.

41. The steps for DNA replication are generally
the same for all prokaryotic and eukaryotic
organisms.
 Unwinding the DNA is accomplished by an
enzyme named DNA helicase.
 Manufacturing new DNA strands is
orchestrated by enzymes called
polymerases.
 Generally, in both prokaryotes and
eukaryotes, the process of DNA replication
proceeds in two opposite directions, from
the origin of replication.

42. Both types of organisms also follow a
pattern called semi-conservative
replication. In this pattern, the individual
strands of DNA are manufactured in
different directions, producing a leading
and a lagging strand.
Lagging strands are created by the
production of small DNA fragments called
Okazaki fragments that are eventually joined
together.
Both types of organisms also begin new

43. Prokaryotes do not have
nucleus and other
membrane-bound
organelles, like
mitochondria, endoplasmic
reticulum, and golgi
bodies. The prokaryotic
DNA is present as a DNA-
protein complex called
nucleoid. The replication
occurs in the cytoplasm of
the cell.
In case of eukaryotes, the
organisms that contain a
membrane-bound nucleus,
the DNA is sequestered
inside the nucleus. Hence,
the nucleus is the site for
DNA replication in
eukaryotes.

44. In prokaryotes, DNA
replication is the first
step of cell division,
which is primarily
through binary fission
or budding.
In eukaryotes, cell division
is a comparatively complex
process, and DNA
replication occurs during
the synthesis (S) phase of
the cell cycle.

45. in prokaryotes, a
single replication site
is present in the
circular DNA molecule.
In eukaryotes, multiple
replication sites are present
in a single DNA molecule.

47. DNA Polymerase I
5' to 3' polymerase, 3' to 5'
exonuclease, 5' to 3'
exonuclease
DNA Polymerase III
5' to 3' polymerase, 3' to 5'
exonuclease

48. DNA polymerase α 5' to 3' polymerase
DNA polymerase δ
5' to 3' polymerase, 3' to 5'
exonuclease
DNA polymerase ε 5' to 3' polymerase

50. One replication bubble
is formed during DNA
replication.
Numerous replication
bubbles are formed in one
replicating DNA molecule.

51. Only two replication
fork formed in each
replicating prokaryotic
chromosome, as DNA
replication is
bidirectional.
A number of replication
forks are formed
simultaneously in each
replicating DNA.

52. Prokaryotic Okazaki
fragments are longer,
with the typical length
observed
in Escherichia coli (E.
coli) being about 1000
to 2000 nucleotides.
The length
of eukaryotic Okazaki
fragments ranges between
100 and 200 nucleotides.
Although comparatively
shorter, they are produced
at a rate slower than that
observed in prokaryotes.

53. In prokaryotes, a
single termination site
is present midway
between the circular
chromosome. The two
replication forks meet
at this site, thus,
halting the replication
process.
In eukaryotes, the linear
DNA molecules have
several termination sites
along the chromosome,
corresponding to each
origin of replication.

55.  Gilbert and Dressier
(1969) described
the rolling circle
model to explain
reactivation in
ssDNA viruses e.g.
Ø × 174 and the
transfer of E. coli
sex factor in
conjugation.

56.  Rolling circle replication describes a process of
unidirectional nucleic acid replication that can rapidly
synthesize multiple copies of circular molecules
of DNA or RNA, such as plasmids,
the genomes of bacteriophages, and the circular
RNA genome of viroids.
 Some eukaryotic viruses also replicate their DNA via
the rolling circle mechanism. Usually under the
name rolling circle amplification, the mechanism is
also widely used in the laboratory in molecular
biology research and in nanotechnology.

57.  Rolling circle has basically five steps:
 DNA will be "nicked".
 The 3' end is elongated using "unnicked"
DNA as leading strand (template); 5' end is
displaced.
 Displaced DNA is a lagging strand and is
made double stranded via a series
of Okazaki fragments.
 Replication of both "unnicked" and displaced
DNA completes.
 Displaced DNA circularizes.

59.  3 basic involve in rolling circular model:
 Initiation
 Elongation
 Termination

60.  Rolling circle DNA replication is initiated by
an initiator protein called Rep-A protein
encoded by the plasmid or bacteriophage
DNA, which nicks one strand of the double-
stranded, circular DNA molecule at a site
called the double-strand origin, or DSO.
 The initiator protein remains bound to the 5'
phosphate end of the nicked strand, and the
free 3' hydroxyl end is released to serve as
a primer for DNA synthesis by DNA
polymerase III.

61.  Producing free 3’-OH and 5’-phosphate
ends, by the action of :
 Helicase
Is in hexameric form. Also
called DNA-b protein.
 Topoisomerase
Use to avoid breakage o the
DNA strand due to stretching.
 Single strand binding protein (SSBPs)
To avoid the reziping of the
DNA strand.

62.  for elongation the DNA polymerase III binds
to the 3’-OH group of broken strand, using
the unbroken strand as a template.
 The polymerase will start move in a circular
form, due to which it is named as Rolling
circular model.
 As the elongation proceeds, the 5’end will
grow out like a waving thread.

63.  Displacement of the nicked strand is carried
out by a host-encoded helicase called PcrA
(plasmid copy reduced) in the presence of
the plasmid replication initiation protein.
 Continued DNA synthesis can produce
multiple single-stranded linear copies of the
original DNA in a continuous head-to-tail
series called a concatemer.

64.  At the point of termination the linear DNA
molecule cleaved from the circle resulting in a
double stranded DNA molecule and a single
stranded linear DNA molecule.
 The linear single stranded molecule is circulized
by the action of Ligase and then replication to
double stranded circular plasmid molecule.

65.  linear copies can be converted to double-stranded
circular molecules through the following process:
 First, the initiator protein makes another nick to
terminate synthesis of the first (leading) strand.
 RNA polymerase and DNA polymerase III then
replicate the single-stranded origin (SSO) DNA to
make another double-stranded circle.
 DNA polymerase I removes the primer, replacing it
with DNA, and DNA ligase joins the ends to make
another molecule of double-stranded circular DNA.

67.  Rolling circle replication has found wide uses in academic
research and biotechnology, and has been successfully
used for amplification of DNA from very small amounts of
starting material.
 Some viruses replicate their DNA in host cells via rolling
circle replication. For instance, human herpesvirus-6 (HHV-
6)(hibv) expresses a set of "early genes" that are believed to
be involved in this process. The long concatemers that
result are subsequently cleaved between the pac-1 and
pac-2 regions of HHV-6's genome by ribozymes when it is
packaged into individual virions.

71. A total of 5 different DNAPs have been reported
in E. coli
 DNAP I: functions in repair and replication
 DNAP II: functions in DNA repair (proven in
1999)
 DNAP III: principal DNA replication enzyme
 DNAP IV: functions in DNA repair (discovered
in 1999)
 DNAP V: functions in DNA repair (discovered
in 1999

72.  DNA Ploymerase α
Synthesis of lagging strand & gap filling
 DNA polymerase β
DNA repair
 DNA polymerase γ
Mitochondrial DNA synthesis
 DNA polymerase δ
Synthesis of leading strand
 DNA polymerase ε
DNA repair
To date, a total of 14 different DNA polymerases have been
reported in eukaryotes

73.  DNA replication is semiconservative
◦ Each strand of template DNA is being copied.
 DNA replication is semidiscontinuous
◦ The leading strand copies continuously
◦ The lagging strand copies in segments (Okazaki
fragments) which must be joined
 DNA replication is bidirectional
◦ Bidirectional replication involves two replication
forks, which move in opposite directions

74.  DNA replication is semiconservative.
the helix must be unwound.
 Most naturally occurring DNA is
slightly negatively supercoiled.
 Torsional strain must be released
 Replication induces positive
supercoiling
 Torsional strain must be released,
again.
 SOLUTION: Topoisomerases

75.  Precedes replicating DNA
 Mechanism
◦ Makes a cut in one strand, passes
other strand through it. Seals gap.
◦ Result: induces positive supercoiling
as strands are separated, allowing
replication machinery to proceed.

76.  Operates in
replication fork
 Separates strands to
allow DNA Pol to
function on single
strands.
Translocate along
single strain in 5’-
>3’ or 3’-> 5’
direction by
hydrolyzing ATP

77. 1. Strand unwinding & separation and formation of replication fork
 (DNA helicase) Helix – unwinding proteins
 Helix – destabilizing proteins--ssb
 Topoisomerase I , II
2. Formation of RNA primer
3. Synthesis of new DNA strands (DNA Pol III)
 Leading strand
 Lagging strand (Okazaki fragments)
4. Excision of RNA primer (DNA Pol I)
5. Sealing the nicks ( DNA ligase)

78.  Replication starts at multiple origins in eukaryotic cells .
 DNA helicases cause unwinding of the duplex DNA from both sides.
 DNA topoisomerases relax the supercoiled DNA ahead of the replication
fork.
 Proceeds in both directions

79.  The two DNA strands are separated
 SSB proteins stabilize the single stranded DNA
 Two replication forks are created
 Each strand acts as a template
 Replication proceeds from 5‘ to 3‘ end

80. Formation of replication fork & SSBs
bind to stabilise ssDNA

81. DNA
helicas
e
DNA helicases unwind
the double helix

83. Limits of DNA polymerase III
 can only build onto 3 end of an
existing DNA strand
5
5
5
5
3
3
3
5
3
5
3 3
Leading strand
Lagging strand
ligase
Okazaki
Leading strand
 continuous synthesis
Lagging strand
 Okazaki fragments
 joined by ligase
 “spot welder” enzyme
DNA polymerase III


3
5
growing
replication fork

84. DNA polymerase III
5
3
5
3
leading strand
lagging strand
leading strand
lagging strandleading strand
5
3
3
5
5
3
5
3
5
3 5
3
growing
replication fork
growing
replication fork
5
5
5
5
5
3
3
5
5
lagging strand
5 3

85. DNA polymerase III
RNA primer
 built by primase
 serves as starter sequence for DNA
polymerase III
Limits of DNA polymerase III
 can only build onto 3 end of an
existing DNA strand
5
5
5
3
3
3
5
3
5
3 5 3
growing
replication fork
primase
RNA

86. DNA polymerase I
 removes sections of RNA primer and
replaces with DNA nucleotides
But DNA polymerase I still
can only build onto 3 end of
an existing DNA strand
5
5
5
5
3
3
3
3
growing
replication fork
DNA polymerase I
RNA
ligase

87. Loss of bases at 5 ends
in every replication
 chromosomes get shorter with each replication
 limit to number of cell divisions?
DNA polymerase III
All DNA polymerases can
only add to 3 end of an
existing DNA strand
5
5
5
5
3
3
3
3
growing
replication fork
DNA polymerase I
RNA
Houston, we
have a problem!

88. Repeating, non-coding sequences at the end
of chromosomes = protective cap
 limit to ~50 cell divisions
Telomerase
 enzyme extends telomeres
 can add DNA bases at 5 end
 different level of activity in different cells
 high in stem cells & cancers -- Why?
telomerase
5
5
5
5
3
3
3
3
growing
replication fork
TTAAGGGTTAAGGG

89. 3’
5’
3’
5’
5’
3’
3’ 5’
helicase
direction of replication
SSB = single-stranded binding proteins
primase
DNA
polymerase III
DNA
polymerase III
DNA
polymerase I
ligase
Okazaki
fragments
leading strand
lagging strand
SSB

90.  DNA polymerase III
◦ 1000 bases/second!
◦ main DNA builder
 DNA polymerase I
◦ 20 bases/second
◦ editing, repair & primer removal
DNA polymerase III
enzyme
Arthur Kornberg
1959
Roger Kornberg
2006

91. RNAse degrades RNA base
paired with DNA
Removal of RNA primers
leaves gaps
DNA polymerase fill the gaps
DNA ligase repairs the
remaining nicks

103.  Occurs @ specific site opposite ori c
 ~350 kb
 Flanked by 6 nearly identical non-palindromic*, 23
bp terminator (ter) sites
 * Significance?
Tus Protein-
arrests
replication fork
motion

104.  Expect 1/103-4, get 1/108-10.
 Factors
◦ 3’5’ exonuclease activity in DNA
pols
◦ Use of “tagged” primers to initiate
synthesis
◦ Battery of repair enzymes
◦ Cells maintain balanced levels of
dNTPs

106. DNA
protein
some
chromosom
al RNA

107. G1
preparing for DNA
replication (cell
growth)
S
DNA replication
G2
a short gap before
mitosis
M
mitosis and cell
division

108. RNA
PROTEIN
DNA
Ceenntrtraall
Dooggma

109. Conservative
replication
Dispersive
replication

110. "Heavy" DNA(15N)
grow in 14N
medium
The first
generation
grow in 14N
medium
The second
generation

111. feature Prokaryote Eukaryote
location Occurs inside the cytoplasm Occurs inside the nucleus
ori one origin of replication per
molecule of DNA
Have many origins of
replication in each
chromosome
Ori length 100-200 or more
nucleotides in length
about 150 nucleotides
Initiation element carried out by protein DnaA
and DnaB
carried out by the Origin
Recognition Complex
DNA polymerase I,II,III α,β,γ,δ,ε

113. •
•
•
•
•
•
•
Semi-conservative
Starts at the ‘origin’
Semi-discontinuous
Synthesis always in the 5'--> 3' direction
RNA primers required
In humans occurs at the rate of 3000
nucleotide/minute.
In bacterial cell it occurs at the rate of 30,000
nucleotide/minute.

114. •
•
•
•
•
•
•
DNA Polymerases 
Helicase
Topoisomerase
Primase
Ligase
SSB Proteins
dNTPs, Mg+2,& ATP
12

116. •
•
Involved in initiation
Synthesizes an RNA primer then adds dNTPs
• A complex of four subunits 50-kD and 60-kD are
primase subunits;180-kD subunit DNA polymerase,
Synthesizes 10-12 nt RNA primers.
• synthesizes RNA primers for the leading strands and
each lagging strand fragments.

117. • The principal DNA polymerase ,add okazaki fragments in
lagging strand.
• Has role in proofreading.
• Consists of a 125 kD and a ~50 kD subunit.
• The 50 kd subunit interacts with PCNA (Proliferating Cell
Nuclear Antigen).

118. •
•
•
Catalyze replication of leading strand ,also
contribute in proofreading.
DNA Polymerase ε Consist of more than one
subunit, >300 kd
Polymerases  and  both contain th 3‘
5'exonuclease activity required for proof reding.
•
•
DNA Polymerase β:
Monomeric having 36-38 kd Involved in DNA repair
process.
DNA Polymerase γ:
The DNA-replicating enzyme of mitochondria

119. PCNA (Proliferating Cell Nuclear Antigen)
Provide substrate for DNA Polymerase δ, the eukaryotic
counterpart of the Sliding Clamp of E. coli PCNA also encircles
the double helix.
RPA (Replication Protein A)
ssDNA-binding protein that facilitates the unwinding of the
helix to create two replication forks ,the eukaryotic counterpart
of the SSB protein.
RFC (Replication Factor C)
The eukaryotic counterpart of the complex Clamp Loader of E.
coli that is it loads PCNA on DNA .

120. ORC ( Origin recognition complex)
Multisubunit protein, binds to sequences within
replicator Interacts with two other proteins – CDC6 &
CDT1 resulting in loading of MCM complex on DNA .
MCM (Mini chromosome maintenance complex)
Heterohexamer(MCM2-MCM7), ring shaped
replicative helicase, Once the Mcm proteins have
been loaded onto the chromatin, ORC and Cdc6 can
be removed from there.
.

121. • They are class of enzymes that catalyze the formation of
alpha phosphodiester bond between two DNA chains.
• This enzyme bind OH group at the 3' end of DNA strand
to phosphate group at 5' end of the other.
• linking the two fragments and completing replication of
the region of the lagging strand.

122. STAGES
Initiation
Elongation
Termination

123. – During initiation, proteins bind to the origin of replication
while helicase unwinds the DNA helix and two replication
forks are formed at the origin of replication.
– During elongation, a primer sequence is added with
complementary RNA nucleotides, which are then replaced
by DNA nucleotides.
– During elongation the leading strand is made continuously,
while the lagging strand is made in pieces called Okazaki
fragments.
– During termination, primers are removed and replaced with
new DNA nucleotides and the backbone is sealed by DNA
ligase.

124. To accomplish this, initiation is partitioned into two
temporally discrete steps:
•Pre replication complex formation during G1 phase
•Activation of pre RC by cdk and ddk enzymes in s phase.

125. •
•
•
•
•
G1phase of the cell cycle there are low levels of CDK
activity.
This allows for the formation of new pre-RC
complexes
During the remaining phases of the cell cycle there
are elevated levels of CDK activity.
This inhibiting new pre-RC complex formation.
At the transition of the G1stage to the S phase of the
cell cycle, S phase–specific cyclin-dependent protein
kinase (CDK) and Dbf4 kinase (DDK) transform the
pre-RC into an active replication fork.

127. • Recognition of the replicator by the
eukaryotic initiator, ORC (Origin
recognition Complex)
• Once ORC is bound, it recruits two helicase
loading proteins Cdc6 (cell division cycle 6
protein) and cdt1 (chromatin licensing and
DNA replication factor 1 protein).
• ORC and the loading proteins recruite
eukaryotic replication protein i.e. helicase
(the Mcm 2-7 complex)
• Instead the pre-RCs that are formed during
G1are only activated to initiate replication
after cells pass from the G1to the S phase of

128. •
proteins at
initiation of
additional replication
the origin and the
replication at S phase.
• These new proteins include the
three eukaryotic DNA polymerases.
• Then converted to the active
CMG(Cdc45-MCM-GINS) helicase
during S phase.
• OnceDNA Polα/ primase
synthesizes an RNA primer and
briefly extends it. Thus initiation
of replication started.

129. RFC ( replication
factor c) recognizes
primer-template
junctions and loads
PCNA ( proliferating
cell nuclear antigen)
at these sites.

131. ahead of•Topoisomerases are responsible for removing supercoils
the replication fork.
•Helicase unwind the strands at fork.
•Pol α is associated with an RNA primase synthesizing a primer of 10
nucleotide stretch of RNA.
•Pol α for priming synthesis & elongates the newly formed primer with
DNA nucleotides.

132. •
•
•
The progress of the replication fork in eukaryotes is
maintain by helicase activity .
The separated polynucleotides are prevented from
reattaching by SSBs Proteins.
The main SSBs Protein in eukaryotes is RPA

134. • Starts with the primase activity of DNA Pol α to lays down an RNA
primer, then the DNA pol component of Pol α adds a stretch of
DNA.
• RFC assembles PCNA at the end of the primer, PCNA displaces
DNA Pol α.
• DNA polymerase δ or e binds to PCNA at the 3’ ends of the
growing strand to carry out highly processive DNA synthesis
Leading strand synthesis.
• The DNA Polymerase α can extend the initial RNA primer with
about 20 nucleotides of DNA but not capable of lengthy DNA
synthesis.
• After that DNA polymerase δ recognizes this primer and begins
leading strand synthesis in 5′ —> 3′ direction,

136. • RNA primers synthesized by DNA polymerase α. in the 5′ 3′
direction.
• DNA polymerase d will synthesize short fragments of DNA called
Okazaki fragments which are added to the 3' end of the primer.
• Completion of lagging-strand synthesis requires removal of the RNA
primer from each okazaki fragments.
• To solve this problem there is two models for completion of lagging-
strand synthesis in eukaryotes are describe:
•
•
The RNase H model
The flap model

137. 36
Rnase H model

139. • Histone syntesis is continuous threw out the cell
cycle but most amount produced during s phase.
• protein responsible for that are NAP-1 (nucleosome
assembly protein) having a role in to transport
histone from cytoplasm to nucleus.
• other protein is CAF-1(chromatin assembly factor)
that deliver histone to the site of replication .

140. TERMINATION

141. • DNA polymarase can not replicate the end of
lagging strand because polymerase requires 3' OH
end to bind at daughter strand. so it require some
additional enzyme to perform it.
•
•
TELOMERE is G - C rich terminal portion of
chromosome.
Highly repetative and conserved sequenced present
in eukaryotes.
•

TELOMERIC sequence in -
MAMMALS- TTAGGG
 ARABIDOPSIS- TTTAGGG
 PARAMECIUM-TTGGGG

142. •
• Telomerase contains protein and RNA which is complementary
to the DNA sequence found in the telomeric repeat.
Telomerase extend the 3' end of the parental chromosome
beyond the 5‘ end of the daughter strand.
• Telomeric repeat-binding factor (TRF1) and TRF2 bind to
double-stranded telomeric DNA and have a role in telomere
stabilization and telomere-length regulation. The protruding 3′
end can invade the duplex DNA and form a lariat-like structure
called ‘t-loop’, establishing a protective cap .

145. • Telomerase activity is repressed in the somatic cells of
multicellular organism, resulting in a gradual shortening of the
chromosomes with each cell generation, and ultimately cell
death (related to cell aging).
• This telomerase activity find in germ cells and absent in
somatic cell, except cancerous cell.
• In humans due to shortening of telomeric length cause a
inherited diseases of premature aging known as progerious
occur in teen age.

146. • It takes E. coli <1 hour to copy
5 million base pairs in its single chromosome .
• Human cell copies its 6 billion bases & divide into daughter cells
in only few hours with remarkably accuracy.
• DNA polymerase initially makes about 1 in 10,000 base pairing
errors .
• Enzymes proofread and correct these mistakes .
• The new error rate for DNA that has been proofread is 1 in 1
billion base pairing errors .

147. • In order to preserve genetic information during cell
division, DNA replication must be completed with
high fidelity.
• To achieve this task, eukaryotic cells have proteins
in place during certain points in the replication
process that are able to detect any errors during
DNA replication and are able to preserve genomic
integrity.
• These checkpoint proteins inactivate cyclin-
dependent kinases to inhibit cell cycle progression
from G1 to S.

148. Protein Function
ATM
ataxia-telangiectasia
mutated
response to DNA damage
& arrest cell cycle
ATR ataxia- and Rad3-related Arrest cell cycle
PRKDC
DNA-dependent protein
kinase catalytic subunit
(DNA-PKcs)
Arrest cell cycle

149. •
•
•
Telomeres are special nucleoprotein structures composed of double-
stranded (TTAGGG)n DNA repetitive sequence ranging from ∼3to 15 kb
and a number of telomere associated proteins.
The ds telomeric DNA terminates at a 3′ single-stranded G-rich
overhang of about 12–500 bases. This protruding 3′ end can invade
the duplex DNA and form a lariat-like structure called ‘t-
loop’.Establishing a protective cap that shields chromosome ends
from being recognized as damaged DNA and prevents nucleolytic
degradation and inappropriate fusions of telomeres.
The t-loop is stabilized by a complex of ds and ss stranded telomere
binding proteins known as the ‘shelterin’ proteins [TRF1, TRF2]. The
primary role of shelterin is to mark telomeres as the natural
chromosome ends and suppress the DNA damage response pathways
at telomeres

150. •
•
•
•
proofreads & corrects mistakes
repairs mismatched bases
removes abnormal bases
repairs damage
throughout life
 Proofreading refers to any mechanism for
correcting errors in protein or nucleic acid
synthesis that involves scrutiny of individual
units after they have been added to the chain
• DNA polymerase  and 

152. •
•
•
•
The process of DNA replication is highly conserved
throughout evolution
Major replication features in simpler organisms
extend uniformly to eukaryotic organisms
Thus eukaryotes replicate their DNA in semi
conservative manner
So, the complete and accurate DNA replication is
integral to the maintenance of the genetic integrity of
all organisms.

155.  Repeated cycles of DNA denaturation,
renaturation with primer oligonucleotide
sequences and replication ,resulting in
exponential growth….
 By using this technique thousand copies of
sample DNA can be produce

156.  PCR Machine
 DNA sample
 Buffer solution
 MgCl2
 Primer F/R
 Taq polymerase
 dNTPs

157.  This includes 3 steps
 1) Denaturation
 2)Annealing
 3)Extention

158.  Denaturation
 94-96 C
 Two strands
of DNA are
separated by
heating DNA
sample
 Annealig
 55 -65 C
 In this step
primer is
attached with
sample DNA
 Extantion
 72C is
 DNA strand is
extended
and dNTPs
are added