Lecture 2: 1
9/11/2006
DNA structure: topics
Topic 1: Structure of the Atom
Topic 2: Chemical Bonds
Topic 3: Primary Structure of DNA
Lecture 2: 2
9/11/2006
DNA structure:
Structure of the Atom/subatomic particles
What is atom?
An atom is the smallest unit of matter, which retains the chemical properties
of that element.
What is an element?
An element is a substance that is made
entirely from one type of atoms.
Electrons
Nucleus
What is the atomic nucleus made of?
It consisting of a dense, central, positively charged nucleus
surrounded by a system of negatively electrons.
The nucleus of atoms is made of protons and neutrons .
except that of hydrogen atoms, whose nucleus has one proton and no neutron.
Protons are positively charged while neutrons has no charge. A neutron has
no electrical charge. It can decay into a proton plus an electron. The number
of protons in the atom is called atomic number.
Lecture 2: 6
9/11/2006
DNA structure:
Structure of the Atom/subatomic particles
What are isotopes?
Isotopes are atoms with the same number of protons, but different
numbers of neutrons, thus different masses. For example, the
hydrogen element has three isotopes:
Protium 1H1 : one proton, no neutron, and a mass of 1 Da;
Deuterium 2H1 : one proton, one neutron and a mass of 2 Da); and
Tritium 3H1 : one proton, two neutrons and a mass of 3 Da
Electrons
Nucleus
What are radioisotopes?
Since excessive neutrons in tritium makes the atoms unstable,
thus cause emission of radiation (beta rays), tritium is referred
to as RADIOATIVE ISOTOPE. The radiation can damage
cells. Tritium has a half-life of 12.32 years
What is atomic weight?
It is equal to the total mass of all subatomic particles including
protons, neutrons and electrons. A single proton weighs 1.660
X 10-24 gram. A neutron with a weight of 1.675 X 10-24
gram is slightly more massive than a proton. The mass of an
electron is 9.10938188 × 10-27 grams. The atomic weight is
usually expressed in Daltons (Da). One Da has the equivalent
mass of one proton.
A teaspoonful of tightly
packed neutrons would
weigh millions of tons
at the earth's surface.
Lecture 2: 7
9/11/2006
DNA structure:
Structure of the Atom/subatomic particles
What are the sizes of atoms?
H
C
N
O
Electrons
1.2 Ǻ
2.0 Ǻ
1.5Ǻ
1.4 Ǻ
Nucleus
Electrons, what you are?
A electron carries a negative charge of 1.60 X 10 -19 Coulomb.
Electrons can be free or orbit around the nucleus of an atom.
An electron is lighter than a proton by 1,836 times.
Electrons in atoms exist in spherical shells of various radii, representing
energy levels. The larger the spherical shell, the higher the energy
contained in the electron.
Lecture 2: 8
9/11/2006
DNA structure:
Chemical Bonds/orbitals
ENERGY LEVEL
Y
Shell
Orbital
Number of electrons
K
1s
2
L
2s
2p
M
3s, 3p, 3d
2
6
18
N
4s, 4p, 4d, 4f
32
Y
X
Z
X
Z
1S
Y
X
Z
PX
Y
X
Z
PY
PZ
What is Fluorescence?
Fluorescence is a luminescence that is mostly found as an optical
phenomenon in cold bodies, in which the molecular absorption of a photon
triggers the emission of another photon with a longer wavelength.
The energy difference between the absorbed and emitted photons ends up as
molecular vibrations or heat. Usually the absorbed photon is in the ultraviolet
range, and the emitted light is in the visible range, but this depends on the
absorbance curve and Stokes shift of the particular fluorophore.
Fluorescence is named after the mineral fluorite, composed of calcium fluoride,
which often exhibits this phenomenon.
What is Fluorescence?
Excitation:
Fluorescence (emission):
hν is a generic term for photon energy where: h = Planck's constant and ν =
frequency of light.
Planck's constant has units of energy multiplied by time, which are the units of
action (J·s).
What is Frequency?
Frequency is the measurement of the number of
times that a repeated event occurs per unit of
time. It is also defined as the rate of change of
phase of a sinusoidal waveform.
Frequency has an inverse relationship to
the concept of wavelength. The frequency
f is equal to the speed v of the wave
divided by the wavelength λ (lambda) of
the wave:
A (aka amplitude) = peak deviation from ceter
Ψ=angular frequency (radians per second)
initial phase (t=0) = -Ψ
Electromagnetic spectrum:
Visible radiation (light)
Above infrared in frequency comes visible light. This is the range in
which the sun and stars similar to it emit most of their radiation. It is
probably not a coincidence that the human eye is sensitive to the
wavelengths that the sun emits most strongly. Visible light (and nearinfrared light) is typically absorbed and emitted by electrons in
molecules and atoms that move from one energy level to another.
The light we see with our eyes is really a very small portion of the
electromagnetic spectrum. A rainbow shows the optical (visible) part
of the electromagnetic spectrum; infrared (if you could see it) would
be located just beyond the red side of the rainbow with ultraviolet
appearing just beyond the violet end.
[edit]
Ultraviolet light
Next in frequency comes ultraviolet (UV). This is radiation whose
wavelength is shorter than the violet end of the visible spectrum.
Being very energetic, UV can break chemical bonds, making
molecules unusually reactive or ionizing them, in general changing
their mutual behavior. Sunburn, for example, is caused by the
disruptive effects of UV radiation on skin cells, which can even cause
skin cancer, if the radiation damages the complex DNA molecules in
the cells (UV radiation is a proven mutagen). The Sun emits a large
amount of UV radiation, which could quickly turn Earth into a barren
desert, but most of it is absorbed by the atmosphere's ozone layer
before reaching the surface.
[edit]
X-rays
After UV come X-rays. Hard X-rays are of shorter wavelengths than
soft X-rays. X-rays are used for seeing through some things and not
others, as well as for high-energy physics and astronomy. Neutron
stars and accretion disks around black holes emit X-rays, which
enable us to study them.
Cdse quanton dots are the newest DNA fluorescent TAGs
A quantum dot is a semiconductor nanostructure that confines the motion of
conduction band electrons, valence band holes, or excitons (pairs of conduction
band electrons and valence band holes) in all three spatial directions. The
confinement can be due to electrostatic potentials (generated by external
electrodes, doping, strain, impurities), due to the presence of an interface between
different semiconductor materials (e.g. in the case of self-assembled quantum dots),
due to the presence of the semiconductor surface (e.g. in the case of a
semiconductor nanocrystal), or to a combination of these. A quantum dot has a
discrete quantized energy spectrum.
Ethidium bromide is an intercalating agent commonly used as a
nucleic acid stain in molecular biology laboratories for techniques such
as agarose gel electrophoresis.
When exposed to ultraviolet light, it will fluoresce with a red-orange
color, intensifying almost 20-fold after binding to DNA. This is probably
not due to rigid stabilization of the phenyl moiety, because the phenyl
ring has been shown to project outside the intercalated bases. The
increased hydrophobicity of the environment is believed to be
responsible.
SYBR is a safer stain for DNA than ethidium bromide
Q- What other excitation wavelengths can I use to visualize SYBR Safe stain?
A - SYBR Safe stain has two main excitation peaks: in the UV at 280 nm, and in the visible region at
502 nm. Thus, 254 nm or 300 nm UV-excitation will work, as will 488 nm lasers, 470 nm LEDs, and
broad blue excitation (such as Invitrogen's Safe Imager (coming September 2005) and Clare
Chemical's Dark Reader). The full excitation and emission spectra for SYBR Safe stain are provided
on our website and in the protocol provided with the stain.
Q - Is SYBR Safe stain really safe? Do I have to use gloves when I use it?
A - In numerous tests carried out by independent, licensed testing laboratories, SYBR
Safe stain showed little or no genotoxicity and no acute toxicity. This stain is not
classified as hazardous waste under U.S. federal regulations; nevertheless, please
exercise common safe laboratory practice when using this reagent.
Biochemistry. 1977 Aug 9;16(16):3647-54.
Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids.
Olmsted J 3rd, Kearns DR.
The mechanism of the enhancement of the fluorescence of ethidium bromide on
binding to double helical RNA and DNA has been investigated. From an
examination of the effect of different solvents on the fluorescence lifetime,
quenching of fluorescence by proton acceptors, and the substantial lengthening
of lifetime observed upon deuteration of the amino protons, regardless of the
medium, we conclude that proton transfer from the excited singlet state is the
process primarily responsible for the approximately equal to 3.5-fold increase in
the lifetime of free ethidium bromide in going from H2O to D2O; the fact that
addition of small amounts of water to nonaqueous solvents decreases the
fluorescence whereas addition of small amounts of D2O enhances the
fluorescence; and the enhancement of the ethidium bromide triplet state yield on
binding to DNA. Other proposed mechanisms are shown to be inconsistent with
our findings.
Stokes shift is the difference (in wavelength or frequency units) between positions of the
band maxima of the absorption and luminescence spectra (or fluorescence) of the same
electronic transition. It is named after Irish physicist George G. Stokes.
When a molecule or atom absorbs light, it enters an excited electronic state. The Stokes
shift occurs because the molecule loses a small amount of the absorbed energy before rereleasing the rest of the energy as luminescence or fluorescence (the called Stokes
fluorescence), depending on the time between the absorption and the reemission. This
energy is often lost as thermal energy.
Green fluorescence Protein:
Excitation maximum: 395 nm
Emisstion maximum: 475 nm
DNA structure:
Structure of the Atom/subatomic particles
Lecture 2: 2
9/11/2006
Lecture 2: 9
9/11/2006
DNA structure:
Chemical Bonds/orbitals
1S
2S
2Px
2Py
2Pz
3S
H1
1S
N7
1S22S22P3
O8
1S22S22P4
C6
1S22S3P3
P15
1S22S22P63S23P3
Na11
Chemical bonds is driven by atoms’ propensity for minimizing the
unpaired electron.
1S22S22P63S
Lecture 2: 10
9/11/2006
DNA structure:
Chemical Bonds/ionic and covalent bonds
Ionic Bonds:
Electrostatic attractions between two
opposite charge ions such as Na+ and
CH- in NaCL (table salt). In this
interaction, sodium atom loses its
electron in the outmost shell to the
chloride atom, becoming positively
charged, while chloride atom receives
an electron from the sodium atom and
turns into a negatively charged ion.
Covalent bonds:
Atoms form a stable linkage by sharing electrons.
Hydrogen gas (H2): H:H
Carbon dioxide (Co2): O:C:O
H
H
C=C
H
H
H20
o
H
H
DNA structure:
Chemical Bonds/Hydrogen bonds
Hydrogen bonds:
A hydrogen is shared by two electroneative
atoms (e.g. N and O) to give a hydrogen
bond.
1 Ǻ long
1 Ǻ long
Lecture 2: 11
9/11/2006
DNA structure:
Chemical bonds/weak interactions
Lecture 2: 12
9/11/2006
Van der Waals forces:
Hydrophobic interaction forces:
Electronstatic attractions or repulsions between
two atoms due to fluctuating electrical charges.
Atoms can be forced to clump together
when non-polar molecules are mixed with
water, which is strongly polar.
DNA structure:
Chemical Bonds/molecules
A molecule is:
the most basic unit of a substance, consisting two or atoms of one or types, bound to
each other by chemical forces. It retains the chemical and physical properties of that
substance.
DNA
is a molecule:
made up of hydrogen, carbon, nitrogen, oxygen, phosphor and metal atoms.
What is a mole of substance?
A mole of substance is the amount of 6.0221415×1023 molecules.
6.0221415×1023 is also called Avogadro's number, named after Amedeo Avogadro.
The value of one mole is equal to the mass in grams given by the formula weight (FW) or
relative molecular weight (Mr). For example, the formula weight of NaCL is 58.44. So, a
mole of sodium chloride weighs 58.44 grams on earth. Carbon has a Mr of 12. That
means 12 grams of carbon contain a mole of carbon.
The concept of mole has been applied to define other particles
such as atoms.
one mole of water is equivalent to about 18 grams of water and contains one mole of H2O
molecules, but three moles of atoms (two moles H and one mole O).
Lecture 2: 14
9/11/2006
DNA structure:
Chemical Bonds/their energy levels
Characteristics of covalent and noncovalent chemical bonds
One calorie:
The quantity of energy need to raise the temperature
of 1 gram of water by 1 oC under one atmosphere
pressure. 1000 calories are one kilocalories (kcal),
which is equal to 4.17 kilojoules (kJ).
Lecture 2: 13
9/11/2006
DNA structure:
Chemical Bonds/molecule weight&mass
Two ways to quantify the amount of a molecule:
molecular weight and molecular mass
Molecular weight (FW, Mr):
the relative ratio of the amount of a mass of a molecule of that
substance to one-twelfth the mass of carbon isotope 12C. The
value of molecular weight is the same as those of the formula
weight (FW) or the relative molecular weight (Mr).
Molecular mass (m):
the total mass of the atoms in a molecule. So, its unit is dalton. One dalton is
equivalent to one-twelfth the mass of carbon-12; a kilodalton (kDa) is 1,000 daltons;
a megadalton (MDa) is 1 million daltons.
What is the difference between molecular weight and molecular
mass?
The former varies with the amount of gravity but the latter does not. Generally, the
two numbers are the same, since substances we deal with are on Earth.
Molar concentration:
One molar denotes one mole (in grams) of a substance (solute) per one liter of a
solvent, expressed in [mole/L). Milimolar: mM; micromolar: µM.
Lecture 2: 15
9/11/2006
Lecture 2: 16
9/11/2006
DNA structure:
Primary Structure of DNA/building blocks
(H, C, O)
ß-D-2-deoxyribose (sugar)
(H, C, O, N)
OH
H
C
5
4
C
H
OH
O
H
H
1
H
3
2
C
OH
C
H
(P, O, ?)
Phosphate
O-O
P
O-
Adenine (a base)
H
H
O
N
C
H
H
N
7
C5
9
N
C4
C
6
C8
H
1N
2
3
N
C
H
Lecture 2: 17
9/11/2006
DNA structure:
Primary Structure of DNA/nucleosides
NH2
Adenine (base)
N
7
C8
H
9
N
ß-D-2-deoxyribose (sugar)
OH
H
C
5
4
C
H
C5
C
6
C4
Sugar + Base = Nucleoside
1N
2
3
N
C
deoxyadenosine
(deoxynucleoside)
H
NH2
H
OH
O
H
H
3
C
OH
H20
1
H
2
C
H
C
H
H
C
5’
4’ C
H
O
H
H
3’
C
OH
C5
9
N
C4
C8
H
OH
N
7
C
6
1N
2
3
N
C
1’
H
2’
C
H
C
H
N-glycosidic
bond
H
DNA structure:
Primary Structure of DNA/two configurations of adenosine
Lecture 2: 18
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Lecture 2: 19
9/11/2006
DNA structure:
Primary Structure of DNA/nucleotides
Phosphate
O-O
O
H
O
H
+
O
P
C
5’
4’ C
H
NH2
H
H
3’
C
OH
C5
9
N
C4
C8
H
O
H
N
7
C
6
1N
2
3
N
C
-H2O
Phosphoester
bond
phosphoester
bond
H
1’
H
2’
C
H
C
N-glycosidic bond
H
Adenosine (nucleoside)
H
Deoxyadenosine
5’-monophosphate
(dAMP)
Adenine deoxynucleotide
Lecture 2: 20
9/11/2006
DNA structure:
Primary Structure of DNA/nucleotides
As in
dAMP
As in
dAMP
As in
dADP
As in
dATP
Lecture 2: 21
9/11/2006
DNA structure:
Primary Structure of DNA/nucleotides
C
Cytosine
deoxynucleotide
DNA structure:
Primary Structure of DNA/nucleotides
Lecture 2: 22
9/11/2006
BASE
DEOXYNUCLEOSIDE
(Base + deoxyribose)
DEPXYNUCLEOTIDE 5’-MONOPHOSPHATE
(Nucleotide)(Base + deoxyribose + phosphate)
Adenine
deoxyadenosine
deoxyadenosine 5’-monophosphate (dAMP)
or adenine nucleotide; A
Guanine
deoxyguanosine
deoxyguanosine 5’-monophosphate (dGMP)
or guanine nucleotide; G
Cytosine
deoxycytidine
deoxycytidine 5’-monophosphate (dCMP)
or cytosine nucleotide; C
Thymine
deoxythymidine
deoxythymidine 5’-monophosphate (dTMP)
or thymine nucleotide; T
Lecture 2: 23
9/11/2006
DNA structure:
Primary Structure of DNA/dinucleotides
DNA is polar
Phosphodiester
linkage
DNA structure:
Primary Structure of DNA/polynucleotide
DNA is a polar, linear polymer made up
of four types of deoxyribose nucleotide
connected by phosphodiester linkage.
Lecture 2: 24
9/11/2006
Lecture 2: 25
9/11/2006
DNA structure:
Primary Structure of DNA/chemical properties of bases
Bases can be present in two tautomeric forms: a keto or its enol form. Two forms
differ in the arrangement of single and double bonds in the rings of purines and
pyrimidines.
Ke
Ke
Tautomerization affects the formation of hydrogen bonds (base-pairing)
DNA structure:
Primary Structure of DNA/base pairing
Lecture 2: 26
9/11/2006
DNA structure:
Secondary Structure of DNA
Lecture 2: 26
9/11/2006
DNA structure:
Primary Structure of DNA/alternative base pairing and modified bases
Lecture 2: 26
9/11/2006
DNA structure:
Primary Structure of DNA/base pairing
Lecture 2: 26
9/11/2006