Topic 1: Structure of the Atom

Topic 2: Chemical Bonds

Topic 3: Primary Structure of DNA

Lecture 2: 19/11/2006

DNA structure: topics

Lecture 2: 29/11/2006

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.

Nucleus

ElectronsWhat is an element?An element is a substance that is made entirely from one type of atoms.

What is the atomic nucleus made of?

The nucleus of atoms is made of protons and neutrons .except that of hydrogen atoms, whose nucleus has one proton and no neutron.

It consisting of a dense, central, positively charged nucleus surrounded by a system of negatively electrons.

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.

Nucleus

Electrons

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); andTritium 3H1 : one proton, two neutrons and a mass of 3 Da

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.

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 are radioisotopes?

A teaspoonful of tightly packed neutrons would weigh millions of tons at the earth's surface.

Lecture 2: 69/11/2006

DNA structure: Structure of the Atom/subatomic particles

Nucleus

Electrons

What are the sizes of atoms?

H C N O

1.2 Ǻ 1.5Ǻ2.0 Ǻ 1.4 Ǻ

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.

Electrons, what you are?

Lecture 2: 79/11/2006

DNA structure: Structure of the Atom/subatomic particles

Lecture 2: 89/11/2006

DNA structure: Chemical Bonds/orbitals

K 1s 2L 2s 2

2p 6M 3s, 3p, 3d 18

N 4s, 4p, 4d, 4f 32

Orbital Number of electronsShell

X

Y

Z

X

Y

Z

X

Y

Z

X

Y

Z

1S PZPYPX

EN

ER

GY

LEV

EL

Fluorescence is a luminescence that is mostly found as an optical phenomenon in cold bodies, in which the molecular absorption of a photontriggers 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 ultravioletrange, 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?

What is Fluorescence?

hν is a generic term for photon energy where: h = Planck's constant and ν = frequency of light.

Excitation:Fluorescence (emission):

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.

A (aka amplitude) = peak deviation from ceterΨ=angular frequency (radians per second) initial phase (t=0) = -Ψ

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:

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 near-infrared 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.

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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.

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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.

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.

SYBR is a safer stain for DNA than ethidium bromide

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 re-releasing 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 nmEmisstion maximum: 475 nm

Lecture 2: 29/11/2006

DNA structure: Structure of the Atom/subatomic particles

H1 1S

1S 2S 2Px 2Py 2Pz

N7 1S22S22P3

O8 1S22S22P4

C6 1S22S3P3

P15 1S22S22P63S23P3

Na11 1S22S22P63S

3S

Lecture 2: 99/11/2006

DNA structure: Chemical Bonds/orbitals

Chemical bonds is driven by atoms’ propensity for minimizing the unpaired electron.

Lecture 2: 109/11/2006

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.

Carbon dioxide (Co2): O:C:O

C=CH

H

H

H

Hydrogen gas (H2): H:H

oH H

H20

Lecture 2: 119/11/2006

DNA structure: Chemical Bonds/Hydrogen bonds

Hydrogen bonds: A hydrogen is shared by two electroneativeatoms (e.g. N and O) to give a hydrogen bond.

1 Ǻ long 1 Ǻ long

Lecture 2: 129/11/2006

DNA structure: Chemical bonds/weak interactions

Van der Waals forces:Electronstatic attractions or repulsions between two atoms due to fluctuating electrical charges.

Hydrophobic interaction forces:Atoms can be forced to clump together when non-polar molecules are mixed with water, which is strongly polar.

Lecture 2: 149/11/2006

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: 139/11/2006

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: 159/11/2006

DNA structure: Chemical Bonds/molecule weight&mass

Molecular weight (FW, Mr):the relative ratio of the amount of a mass of a molecule of thatsubstance 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).

Two ways to quantify the amount of a molecule:molecular weight and molecular mass

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.

C C

CC

O

HOH

OH

HH

C

HH

HH1

23

4

5

ß-D-2-deoxyribose (sugar)

C

N

C

N

C

C

N

N

C

N

H

HH

H

H1

23

4

56

78

9

Adenine (a base)

Lecture 2: 169/11/2006

DNA structure: Primary Structure of DNA/building blocks

(H, C, O)

(H, C, O, N)

(P, O, ?)

OH

P

O-

O-O

O-

Phosphate

Lecture 2: 179/11/2006

DNA structure: Primary Structure of DNA/nucleosides

C

N

C

N

C

C

N

N

C

NH2

H

H

H1

23

4

56

78

9

C C

CC

O

HOH

OH

HH

C

HH

HH1

23

4

5

OHC

N

C

N

C

C

N

N

C

NH2

H

H1

23

4

56

78

9

C C

CC

O

HOH

HH

C

HH

HH1’

2’3’

4’

5’

OH

H20

N-glycosidicbond

Adenine (base)

ß-D-2-deoxyribose (sugar)

deoxyadenosine(deoxynucleoside)

Sugar + Base = Nucleoside

Lecture 2: 189/11/2006

DNA structure: Primary Structure of DNA/two configurations of adenosine

Lecture 2: 199/11/2006

DNA structure: Primary Structure of DNA/nucleotides

+P

O-

O-O

O H

Phosphate

C

N

C

N

C

C

N

N

C

NH2

H

H1

23

4

56

78

9

C C

CC

O

HOH

HH

C

HH

HH1’

2’3’

4’

5’

O H

N-glycosidic bond

Adenosine (nucleoside)

-H2O phosphoesterbond

H

Deoxyadenosine5’-monophosphate

(dAMP)

Phosphoesterbond

Adenine deoxynucleotide

As in dAMP

Lecture 2: 209/11/2006

DNA structure: Primary Structure of DNA/nucleotides

As in dAMP

As in dADP

As in dATP

C

Lecture 2: 219/11/2006

DNA structure: Primary Structure of DNA/nucleotides

Cytosine deoxynucleotide

Lecture 2: 229/11/2006

DNA structure: Primary Structure of DNA/nucleotides

BASE

Adenine

Guanine

Cytosine

Thymine

DEOXYNUCLEOSIDE(Base + deoxyribose)

deoxyadenosine

deoxyguanosine

deoxycytidine

deoxythymidine

DEPXYNUCLEOTIDE 5’-MONOPHOSPHATE(Nucleotide)(Base + deoxyribose + phosphate)

deoxyadenosine 5’-monophosphate (dAMP)or adenine nucleotide; A

deoxyguanosine 5’-monophosphate (dGMP)or guanine nucleotide; G

deoxycytidine 5’-monophosphate (dCMP)or cytosine nucleotide; C

deoxythymidine 5’-monophosphate (dTMP)or thymine nucleotide; T

Lecture 2: 239/11/2006

DNA structure: Primary Structure of DNA/dinucleotides

Phosphodiesterlinkage

DNA is polar

Lecture 2: 249/11/2006

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: 259/11/2006

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)

Lecture 2: 269/11/2006

DNA structure: Primary Structure of DNA/base pairing

Lecture 2: 269/11/2006

DNA structure: Secondary Structure of DNA

Lecture 2: 269/11/2006

DNA structure: Primary Structure of DNA/alternative base pairing and modified bases

Lecture 2: 269/11/2006

DNA structure: Primary Structure of DNA/base pairing