Replication
Qui ckTime™ and a GIF decompressor are needed to see thi s pi ctur e.
CH3
N
R
O
H
H N
U
T N H
N
H
N H
R
C N
O
A
N
N
O
N
N
O
H N
H N
H
R
N
G
N
N
R
DeoxyAdenine (dA)
DeoxyAdenineTriphosphate (dATP)
O O O
N
O P O P O P OH
O O O CH2 O
OH
Ribose
DeoxyRibose
DiDeoxyRibose
OH
OH
H
N H
N
A
N
N
5’P
3’OH
5’P 3’OH
3’OH
3’OH 5’P
5’P
3’
3’
3’
Just adding
polymerase
clamp
and
polymerase
clamp loader,
clamp, and
polymerase
clamp loader,
clamp, and
polymerase
primase,
clamp loader,
clamp, and
polymerase
helicase
helicase,
clamp loader,
clamp, and
polymerase
How much is 1010?
Typical single space typewritten page 3000 characters/sheet
1 sheet/3000 characters * 1010 characters = 3,333,333 sheets
1 ream/500 sheets * 3,333,333 sheets = 6666 reams
1 box/10 reams * 6666 reams = 666 boxes
666 boxes of single-spaced typed sheets would fill the front of
this room up to the ceiling with only a single spelling error.
How fast is the fork going?
E. coli can replicate in about 20 minutes under optimal conditions.
E. coli genome contains 4.5*106 basepairs
4.5*106 basepairs/2 replication forks/1200 s =
1875 basepairs/replication fork/s
By comparison….
1 deck of cards/26 pairs
1875 pairs * 1 deck of cards/26 pairs = 72 decks of cards
To move as fast as a replication fork you would have to be able to
sort 72 decks of shuffled cards….
pairing every club with a spade and every heart with a diamond…
each second.
How many mistakes are made each time the cell replicates?
E. coli genome 4.5*106 basepairs
(1 genome/4.5*106 basepairs) * (1*1010 basepairs/1 error)=
2222 genomes/error
H. sapiens genome 3.1*109 basepairs
(1 genome/ 3.1*109 basepairs) * (1*1010 basepairs/1 error)=
3 genomes/error
What would happen in E. coli if mismatch repair did not occur?
What would happen in humans if mismatch repair did not occur?
Sorting it out
And keeping things
straight
2) Supercoiling helps
to condense and pack
the DNA, keeping it
organized so that it
can fit in the cell.
1) Partitioning
functions help to
keep things
sorted/separated as
replication occurs so
that things aren’t too
badly tangled when
they finish.
Tools for examining / rearranging / changing the DNA
Nucleic Acid Hybridization
• DNA denaturation: Two DNA strands can be separated by heat
without breaking phosphodiester bonds
• DNA renaturation = hybridization: Two single strands that are
complementary or nearly complementary in sequence can come
together to form a different double helix
• Single strands of DNA can also hybridize complementary
sequences of RNA
34
Fig. 6.24
Restriction Enzymes
• Restriction enzymes cleave duplex DNA at particular
nucleotide sequences
• The nucleotide sequence recognized for cleavage by a
restriction enzyme is called the restriction site of the enzyme
• In virtually all cases, the restriction site of a restriction
enzyme reads the same on both strands A DNA sequence with
this type of symmetry is called a palindrome
36
Fig. 6.26
Southern Blot Analysis
• DNA fragments on a gel can often be visualized by staining with ethidium
bromide, a dye which binds DNA
• Particular DNA fragments can be isolated by cutting out the small region of
the gel that contains the fragment and removing the DNA from the gel.
• Specific DNA fragments are identified by hybridization with a probe = a
radioactive fragment of DNA or RNA
• Southern blot analysis is used to detect very small amounts of DNA or to
identify a particular DNA band by DNA-DNA or DNA-RNA hybridization
39
Southern Blot Analysis
Fig. 6.27
Polymerase Chain Reaction
• Polymerase Chain Reaction (PCR) makes possible the
amplification of a particular DNA fragment
• Oligonucleotide primers that are complementary to the ends
of the target sequence are used in repeated round of
denaturation, annealing, and DNA replication
• The number of copies of the target sequence doubles in each
round of replication, eventually overwhelming any other
sequences that may be present
41
Polymerase Chain Reaction
• Special DNA polymerase is used in PCR = Taq
polymerase isolated from bacterial thermophiles which
can withstand high temperature used in procedure
• PCR accomplishes the rapid
production of large
amounts of target DNA which can then be identified and
analyzed
42
Polymerase
chain
reaction
(PCR)
Heat
1)
Needs only the
smallest amount of
DNA
2) Short DNA primers
(that you can
synthesize)
Cool
Polymerize
Allows you to
amplify
(generate a ton
of) any gene or
sequence that
you need
DNA Sequence Analysis
•
DNA sequence analysis
determines the order of bases in
DNA
•
The dideoxy sequencing method
employs DNA synthesis in the
presence of small amounts of
fluorescently labeled nucleotides
that contain the sugar
dideoxyribose instead of
deoxyribose
Fig. 6.29
45
DNA Sequencing:
Dideoxy Method
•
Modified sugars cause chain termination because
it lacks the 3’-OH group, which is essential for
attachment of the next nucleotide in a growing
DNA strand
•
The products of DNA synthesis are then
separated by electrophoresis. In principle, the
sequence can be read directly from the gel
DNA Sequencing: Dideoxy Method
•
•
•
•
Each band on the gel is one base longer than the previous band
Each didyoxynucleotide is labeled by different fluorescent dye
G, black; A, green; T, red; C, purple
As each band comes off the bottom of the gel, the fluorescent dye that it
contains is excited by laser light, and the color of the fluorescence is
read automatically by a photocell and recorded in a computer
47
Fig. 6.31