DNA structure:
Non-helical secondary structures: non-helical , palindrome, hairpin, and cruciform
Lecture 9: 1
10/11/2006
stabilized by negative DNA
supercoiling is a cruciform in
which an inverted repeat
nucleotide sequence rearranges
from a fully double-stranded
structure into two base-paired
hairpins.
Images of 106 bp inverted repeat
generated by atomic force microscope.
(i) low salt and low superhelicity;
(ii) (ii) high Salt and high superhelicity.
DNA structure:
Tertiary Structure: Plasmid
Lecture 8: 2
10/11/2006
Primary features of plasmids:
1) extrachromosomal DNA;
2) capable of autonomous replication.
Some other common features:
1. It is typically circular and double-stranded. It usually occurs in
bacteria, sometimes in eukaryotic organisms (e.g., the 2-micrometrering in Saccharomyces cerevisiae).
2. Size of plasmids varies from 1 to over 400 kilobase pairs (kbp).
3. There may be one copy, for large plasmids, to hundreds of copies of
the same plasmid in a single cell, or even thousands of copies.
stringent - low copy number (F factor); relaxed - high copy number
(pBR322 - 16 copies; pUC - 30 to 50 copies)
4. Some plasmids carry antibiotic resistance gene (s) or reporter genes.
5. Multiple cloning sites are present in commercial plasmid vectors.
6. The term plasmid was first introduced by the American molecular
biologist Joshua Lederberg in 1952.
DNA structure:
Tertiary Structure: topology of plasmids
TEM images of various
Forms of a plasmid
Negative and positive
superhelical tension occur when
the helix is unwound or over
wound, respectively
In the cells, plasmids are
negatively supercoiled, thus
favoring the melting and
interactions with proteins.
Lecture 7: 3
10/11/2006
DNA structure:
Tertiary Structure: topology of plasmids
Lecture 7: 4
10/11/2006
DNA structure:
Tertiary Structure: topology of plasmids
Basics of Optics
the relation between focal length f,
object distance a, and image
distance b which is called the lens
equation:
1/a+1/b=1/f
Lens aberrations
(a) spherical
aberration, (b)
chromatic
aberration, (c)
astigmatism, and
(d) coma.
Lecture 7: 5
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The distance from the center of the convex lens to
the focal plane is know as the focal distance. (For
an idealized symmetrical thin convex lens, this
distance is the same in front of or behind the
lens.) The image of our giraffe now appears at the
focal plane (as illustrated in Figure 2). The image
is smaller than the object (the giraffe); it is inverted
and is a real image capable of being captured on
film. This is the case for the camera used for
ordinary scenic photography.
The object is now moved closer to the front of the
lens but is still more than two focal lengths in front of
the lens (this scenario is addressed in Figure 3).
Now, the image is found further behind the lens. It is
larger than the one described above, but is still
smaller than the object. The image is inverted, and is
a real image. This is the case for ordinary portrait
photography.
The object is brought to twice the focal distance in front of the lens. The
image is now two focal lengths behind the lens as illustrated in Figure 4.
It is the same size as the object; it is real and inverted.
The object is now situated between one and two focal lengths in front of the
lens (shown in Figure 5). Now the image is still further away from the back of
the lens. This time, the image is magnified and is larger than the object; it is
still inverted and it is real. This case describes the functioning of all finite tube
length objectives used in microscopy. Such finite tube length objectives project
a real, inverted, and magnified image into the body tube of the microscope.
This image comes into focus at the plane of the fixed diaphragm in the
eyepiece. The distance from the back focal plane of the objective (not
necessarily its back lens) to the plane of the fixed diaphragm of the eyepiece is
known as the optical tube length of the objective.
DNA structure: Tertiary Structure: topology of plasmids
Basics of Optics
In the last case, the object is situated at the front focal plane of the convex
lens. In this case, the rays of light emerge from the lens in parallel. The
image is located on the same side of the lens as the object, and it appears
upright (see Figure 1). The image is a virtual image and appears as if it
were 10 inches from the eye, similar to the functioning of a simple
magnifying glass; the magnification factor depends on the curvature of the
lens.
The last case listed above describes the functioning of the observation
eyepiece of the microscope. The "object" examined by the eyepiece is the
magnified, inverted, real image projected by the objective. When the
human eye is placed above the eyepiece, the lens and cornea of the eye
"look" at this secondarily magnified virtual image and see this virtual image
as if it were 10 inches from the eye, near the base of the microscope.
Since the image appears to be on the same side of the
lens as the object, it cannot be projected onto a screen.
Such images are termed virtual images and they appear
upright, not inverted. Figure 1 presents an illustration of
how a simple magnifying lens operates. The object (in this
case the subject is a rose) is being viewed with a simple
bi-convex lens. Light reflected from the rose enters the
lens in straight lines as illustrated in Figure 1. This light is
refracted and focused by the lens to produce a virtual
image on the retina. The image of the rose is magnified
because we perceive the actual size of the object (the
rose) to be at infinity because our eyes trace the light rays
back in straight lines to the virtual image (Figure 1).
Lecture 7: 7
10/11/2006
DNA structure:
Tertiary Structure: topology of plasmids
Lecture 7: 8
10/11/2006
Basics of Optics
Multi-lens imaging systems achieve very high magnification.
Lecture 7: 9
10/11/2006
DNA structure: Tertiary Structure: topology of plasmids
Bragg's Law refers to the
simple equation:
nλ = 2d sinθ
Bragg's Law can easily be derived by considering the conditions
necessary to make the phases of the beams coincide when the
incident angle equals and reflecting angle. The rays of the incident
beam are always in phase and parallel up to the point at which the
top beam strikes the top layer at atom z (Fig. 1). The second beam
continues to the next layer where it is scattered by atom B. The
second beam must travel the extra distance AB + BC if the two
beams are to continue traveling adjacent and parallel. This extra
distance must be an integral (n) multiple of the wavelength (l) for the
phases of the two beams to be the same:
nλ = AB +BC (2).
Fig. 1 Deriving Bragg's Law using the reflection
geometry and applying trigonometry. The lower beam
must travel the extra distance (AB + BC) to continue
traveling parallel and adjacent to the top beam.
Recognizing d as the hypotenuse of the right triangle
Abz, we can use trigonometry to relate d and θ to the
distance (AB + BC). The distance AB is opposite q so,
AB = d sinθ (3).
Because AB = BC eq. (2) becomes,
nλ = 2AB (4)
Substituting eq. (3) in eq. (4) we have,
nl = 2 d sinθ, (1)
and Bragg's Law has been derived. The location of the
surface does not change the derivation of Bragg's Law.
DNA structure: Tertiary Structure: topology of plasmids
Microscopy
Lecture 7: 10
10/11/2006
The resolution of an optical microscope is defined as the shortest distance
between two points on a specimen that can still be distinguished by the
observer or camera system as separate entities.
There are several equations that have been
derived to express the relationship between
numerical aperture, wavelength, and resolution:
Resolution (r) = λ/(2NA)
(1)
Resolution (r) = 0.61λ/NA
(2)
Resolution (r) = 1.22λ/(NA(obj) + NA(cond)) (3)
Where r is resolution (the smallest resolvable
distance between two objects), NA is a general
term for the microscope numerical aperture, is
the imaging wavelength, NA(obj) equals the
objective numerical aperture, and NA(cond) is
the condenser numerical aperture. Notice that
equation (1) and (2) differ by the multiplication
factor, which is 0.5 for equation (1) and 0.61 for
equation (2).
DNA structure: Tertiary Structure: topology of plasmids
Microscopy
Lecture 7: 11
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Numerical Aperture
Numerical Aperture (also termed Object-Side Aperture) is a value (often
symbolized by the abbreviation NA) originally defined by Abbe for
microscope objectives and condensers. It is given by the simple
expression:
Numerical Aperture (NA) = n • sin(α ) or n • sin(θ)
Note: Many authors use the variable m to designate the one-half angular
aperture while others employ the more common term α , and in some
instances, θ.
In the numerical aperture equation, n represents the refractive index of
the medium between the objective front lens and the specimen, and m or
a is the one-half angular aperture of the objective.
DNA structure: Tertiary Structure: topology of plasmids
Microscopy
Magnifi
4x
10x
20x
40x
60x
100x
Plan Achromat
N.A.
R
0.10
2.75
0.25
1.10
0.40
0.69
0.65
0.42
0.75
0.37
1.25
0.22
N.A. = Numerical Aperture
R=reolution (microm)
Wavelength (nm)
Resolution(μM)
360
400
450
500
550
600
650
700
.19
.21
.24
.26
.29
.32
.34
.37
Objective types
Plan Fluorite
N.A.
R
0.13
2.12
0.30
0.92
0.50
0.55
0.75
0.37
0.85
0.32
1.30
0.21
Lecture 7: 12
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Plan Apochromat
N.A.
R
0.20
1.375
0.45
0.61
0.75
0.37
0.95
0.29
0.95
0.29
1.40
0.20
DNA structure: Tertiary Structure: topology of plasmids
Microscopy
Lecture 7: 13
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DNA structure:
Tertiary Structure: topology of plasmids
Lecture 7: 14
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Basics of magnetic lens
A strong rotationally symmetric, magnetic field between a pair of
cylindrical pole pieces drives paraxially moving particles along a spiral
path that converges towards the axis. This results from magnetic field
components perpendicular as well as parallel to the optical axis (Fig.a).
Thus, a magnetic field of this kind serves as a lens for charged particle
beams.
Rotationally symmetric electrostatic fields are created between cylindrical
electrodes. Trajec tories of charged particles are bent as a result of the
electric field and converge towards a point on the optical axis. (Fig. b).
Lecture 7: 15
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DNA structure:
Tertiary Structure: topology of plasmids
Comparison of two types of electron microscopes
objective
projective
objective
Lecture 7: 16
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DNA structure:
Tertiary Structure: topology of plasmids
Negative staining of biological molecules
for electron microscopy
1) Macromolecules or supramolecular
assemblies are adsorbed from their
suspension onto a thin carbon
support film;
2. Washed with an aqueous solution
of a heavy metal salt (e.g., 1% uranyl
acetate or 2% Na-phosphotungstate),
before the sample is allowed to dry. A
very thin film of metal salt now covers
the support film everywhere except
where it has been excluded by the
presence of an adsorbed
macromolecule.
3. Observe under TEV. Because the
macromolecule allows the electrons
to pass much more readily than does
the surrounding heavy metal film, a
reversed or negative image of the
molecule is created, hence the name
“negative staining”
DNA structure:
Tertiary Structure: topology of plasmids
Agarose Gel Analysis of Biological Molecules (e. g. DNA)
Lecture 7: 17
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DNA structure:
Tertiary Structure: topology of plasmids
Lecture 7: 18
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Chemical and physical properties of agarose
Agar is an unbranched polysaccharide obtained from the cell walls of some species of red
algae or seaweed. The word agar comes from the Malay word agar-agar (meaning jelly). It is
also known as kanten or agal-agal (Ceylon agar). Chemically, agar is a polymer made up of
subunits of the sugar galactose. Agar polysaccharides serve as the primary structural
support for the algae's cell walls. Dissolved in hot water and cooled, agar becomes
gelatinous. Its chief use is as a culture medium for microbiological work. Other uses are as
a laxative, a vegetarian gelatin substitute, a thickener for soups, in jellies, ice cream and
Japanese desserts such as anmitsu, as a clarifying agent in brewing, and for paper sizing
fabrics.
Typical Properties
- Gelling temp : 26º-30ºC
- Melting temp : ≤65ºC
- Moisture content : ≤10%
- Sulfate : ≤0.10%
- EEO, (-mr) : ≤0.10
- Gel strength : ≥200 g/cm2
- RNase/Dnase Activity : None Detected
DNA structure:
Tertiary Structure: topology of plasmids
Properties of Agarose
Lecture 7: 19
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G. parvispora or G. tikvahiae
Source of agarose
Agarose is derived from a series of naturally occurring derivatives from seaweed.
Most agar comes from various species of Gelidium and Gracilaria. All species
contain ester sulfates and some, except Gracilaria, contain varying amounts of
pyruvates. Gracilaria agarose contains methyl ethers, the position of which is
variable according to the species.
Structure of agarose
Agarose consists of 1,3-linked ß-D-galactopyranose and 1,4-linked
3,6-anhydro-α-L-galactopyranose. This basic agarobiose repeat unit forms long
chains with an average molecular mass of 120,000 daltons, representing about 400
agarobiose units. There are also charged groups present on the polysaccharide,
most notably pyruvates and sulfates.
DNA structure:
Tertiary Structure: topology of plasmids
Properties of Agarose
Lecture 7: 20
10/11/2006
Electroendosmosis (EEO)
Electroendosmosis (EEO) is a functional measure of the number of sulfate and
pyruvate residues present on the agarose polysaccharide. This phenomenon occurs
during electrophoresis when the anticonvective medium (the agarose in this case)
has a fixed negative charge. In an electric field, the hydrated positive ions
associated with the fixed anionic groups in the agarose gel migrate toward the
cathode. Water is thus pulled along with the positive ions, and migration of
negative molecules such as DNA is retarded.
How EEO is measured
Electroendosmosis is quantitated by subjecting a mixture of dextran and
albumin to electrophoresis, then visualizing them and measuring their
respective distances from the origin. The amount of EEO expressed in terms
of relative mobility (-mr) is calculated by dividing the migration distance of the
neutral dextran (OD) by the sum of the migration distances of the dextran and
the albumin (OD + OA): -mr = OD/(OD + OA).
DNA structure:
Tertiary Structure: topology of plasmids
Lecture 7: 21
10/11/2006
Properties of Agarose
Gelation
The mechanism for gelation of agarose was first suggested by Rees and later
demonstrated by Arnott. It involves a shift from a random coil in solution to a
double helix in the initial stages of gelation, and then to bundles of double
helices at the final stage. The average pore size varies with concentration and
type of agarose, but is typically 100 to 300 nm.
DNA structure:
Tertiary Structure: topology of plasmids
Properties of Agarose
Lecture 7: 22
10/11/2006
Methylation of agarose
The agarose polysaccharide also contains uncharged methyl groups.
The extent of natural methylation is directly proportional to the gelling temperature.
Unexpectedly, synthetically methylated agaroses have lower, rather than higher,
gelling temperatures, and the degree of synthetic methylation is inversely
proportional to the gelling temperature.