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Structures of nucleic acids II
Southern blot-hybridizations
Sequencing
Supercoiling: Twisting, Writhing
and Linking number
Southern blot-hybridizations
• Allows the detection of a particular DNA sequence
among the many displayed on an electrophoretic
gel.
• e.g. determine which among many restriction
fragments contains a gene.
• Transfer the size-separated DNA fragments out of
the agarose gel and onto a membrane (nylon or
nitrocellulose) to make an immobilized replica of
the gel pattern.
• Hybridize the membrane to a specific, labeled
nucleic acid probe and determine which DNA
fragments contain that labeled sequence.
Steps in Southern blot-hybridization
nylon or
nitrocellulose
membrane
Separate restriction
fragments
by size using
electrophoresis
through an agarose gel.
Denature duplex DNA
Transfer denatured DNA
fragments onto
a membrane to produce an
immobilized replica of the
gel-separated fragments
(the "blot").
Steps in Southern blot-hybridization, continued
+
** *
* *
* **
*
Incubate the blot (with denatured DNA
fragments immobilized) with an
excess of labeled DNA from a specific
gene or region under conditions that
favor formation of specific hybrids.
*
*
*
Wash off any
nonspecifically
bound
probe.
The probe has now
hybridized to the
restriction fragments
that have the gene of
interest.
*
*
*
This pattern of
hybridization to the blot
can be detected by
exposure to X-ray film or
phosphor screens.
Image on the exposed film after developing.
This tells you which restriction fragments
have the gene or region of interest (if you
remembered to include size markers!).
Strategy to determine DNA or RNA sequence
• Generate a nested set of fragments with one
common, labeled end
• The other end terminates at one of the 4
nucleotides
• Electrophoretic resolution of the fragments
allows the reading of the sequence:
Fragment of length 47 ends at G
48
A
49
T
Sequence is …GAT….
Common sequencing techniques
Technique Common end
DNA:
Restriction
Maxam endonuclease
& Gilbert
DNA:
Sanger
Primer for
DNA
polymerase
RNA
Natural
end of
RNA
Label
32P
Nt-specific end
Base-specific
chemical
cleavage
32P
or
Chain termination
fluores- by dideoxycence nucleotides
32P
Nucleotide-specific
enzymatic cleavage
Example of dideoxynucleotide sequencing
• Reactions: Fig. 2.30
• Output: Fig. 2.31
• Cycle Sequencing Movie:
– http://vector.cshl.org/resources/BiologyAnimationLibrary.htm
• The Sanger dideoxynucleotide method is
amenable to automation performed by robots.
• This approach is the one adapted for virtually all
the whole-genome sequencing projects.
Example of output from automated
dideoxysequencing
Supercoiling of topologically
constrained DNA
• Topologically closed DNA can be circular
(covalently closed circles) or loops that are
constrained at the base
• The coiling (or wrapping) of duplex DNA
around its own axis is called supercoiling.
Different topological forms of DNA
Genes VI : Figure 5-9
Negative and positive supercoils
• Negative supercoils twist the DNA about its axis in
the opposite direction from the clockwise turns of
the right-handed (R-H) double helix.
– Underwound (favors unwinding of duplex).
– Has right-handed supercoil turns.
• Positive supercoils twist the DNA in the same
direction as the turns of the R-H double helix.
– Overwound (helix is wound more tightly).
– Has left-handed supercoil turns.
Components of DNA Topology : Twist
• The clockwise turns of R-H double helix
generate a positive Twist (T).
• The counterclockwise turns of L-H helix (Z
form) generate a negative T.
• T = Twisting Number
B form DNA: + (# bp/10 bp per twist)
A form NA: + (# bp/11 bp per twist)
Z DNA: - (# bp/12 bp per twist)
Components of DNA Topology : Writhe
• W = Writhing Number
• Refers to the turning of the axis of the
DNA duplex in space
• Number of times the duplex DNA
crosses over itself
Relaxed molecule W=0
Negative supercoils, W is negative
Positive supercoils, W is positive
Components of DNA Topology : Linking number
• L = Linking Number = total number of times
one strand of the double helix (of a closed
molecule) encircles (or links) the other.
• L=W+T
L cannot change unless one or both
strands are broken and reformed
• A change in the linking number, DL, is
partitioned between T and W, i.e.
•
• if
DL=DW+DT
DL = 0, then DW= -DT
Relationship between supercoiling and twisting
Figure from M. Gellert; Kornberg and Baker
DNA in most cells is negatively supercoiled
• The superhelical density is simply the
number of superhelical (S.H.) turns per turn
(or twist) of double helix.
• Superhelical density = s = W/T = -0.05 for
natural bacterial DNA
– i.e., in bacterial DNA, there is 1 negative
S.H. turn per 200 bp
• (calculated from 1 negative S.H. turn per 20
twists = 1 negative S.H. turn per 200 bp)
Negatively supercoiled DNA favors
unwinding
• Negative supercoiled DNA has energy
stored that favors unwinding, or a transition
from B-form to Z DNA.
• For s = -0.05, DG=-9 Kcal/mole favoring
unwinding
Thus negative supercoiling could favor
initiation of transcription and initiation of
replication.
Topoisomerase I
• Topoisomerases: catalyze a change in the
Linking Number of DNA
• Topo I = nicking-closing enzyme, can relax
positive or negative supercoiled DNA
• Makes a transient break in 1 strand
• E. coli Topo I specifically relaxes negatively
supercoiled DNA. Calf thymus Topo I
works on both negatively and positively
supercoiled DNA.
Topoisomerase I: nicking & closing
One strand passes through a nick in the other strand.
Genes VI : Figure 17-15
Topoisomerase II
• Topo II = gyrase
• Uses the energy of ATP hydrolysis to
introduce negative supercoils
• Its mechanism of action is to make a
transient double strand break, pass a
duplex DNA through the break, and then reseal the break.
TopoII: double strand break and passage