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

Excitation Maxima: 494-502 nMEmission Maxima: 521 nM

SYBR Green I

Stokes shift of SYBR GREEN I

Nucleic Acids Res. 2004 Jul 12;32(12):e103.

Investigations on DNA intercalation and surface binding by SYBR Green I, itsstructure determination and methodological implications.

Zipper H, Brunner H, Bernhagen J, Vitzthum F.

Laboratory of Biochemistry, Institute for Interfacial Engineering, University ofStuttgart, 70569 Stuttgart, Germany.

The detection of double-stranded (ds) DNA by SYBR Green I (SG) is important in many molecular biology methods including gel electrophoresis, dsDNA quantification in solution and real-time PCR. Biophysical studies at defineddye/base pair ratios (dbprs) were used to determine the structure-property relationships that affect methods applying SG. These studies revealed the occurrence of intercalation, followed by surface binding at dbprs above approximately 0.15. Only the latter led to a significant increase in fluorescence. Studies with poly(dA)* poly(dT) and poly(dG)* poly(dC) homopolymers showed sequence-specific binding of SG. Also, salts had a marked impact on SG fluorescence. We also noted binding of SG to single-stranded (ss) DNA, although SG/ssDNA fluorescence was at least approximately 11-fold lower than with dsDNA. To perform these studies, we determined the structure of SG by mass spectrometry and NMR analysis to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3- thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]. For comparison, the structure of PicoGreen (PG) was also determined and is [2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thia zol-2-yl)-methylidene]-1-phenyl quinolinium]+. These structure-property relationships help in the design of methods that use SG, in particular dsDNAquantification in solution and real-time PCR.

Lecture 5: 19/26/2006

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 5: 29/26/2006

DNA structure: Chemical Bonds/orbitals

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

Lecture 5: 39/26/2006

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 5: 49/26/2006

DNA structure: Chemical Bonds/Hydrogen bonds

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

1 Ǻ long 1 Ǻ long

Lecture 5: 59/26/2006

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 5: 69/26/2006

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 5: 79/26/2006

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 5: 89/26/2006

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 5: 99/26/2006

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 5: 109/26/2006

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 5: 119/26/2006

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

H HSyn-deoxyadenosine Syn-deoxyadenosine

Lecture 5: 129/26/2006

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

deoxyadenosine(deoxynucleoside)

-H2O phosphoesterbond

H

Deoxyadenosine5’-monophosphate

(dAMP)

Phosphoesterbond

Adenine deoxynucleotide

Hdeoxyribose

As in dAMP

Lecture 5: 139/26/2006

DNA structure: Primary Structure of DNA/nucleotides

As in dAMP

As in dADP

As in dATP

C

Lecture 5: 149/26/2006

DNA structure: Primary Structure of DNA/nucleotides

Cytosine deoxynucleotide

H

Lecture 5: 159/26/2006

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 5: 159/26/2006

DNA structure: Primary Structure of DNA/dinucleotides

Phosphodiesterlinkage

DNA is polar

Lecture 5: 179/26/2006

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 5: 189/26/2006

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 5: 199/26/2006

DNA structure: Primary Structure of DNA/base pairing

Lecture 5: 209/26/2006

DNA structure: Secondary Structure of DNA

Lecture 5: 219/26/2006

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

AAAAACAAGAAAATGATATAGAAAAAACAGTGGTCAATCAATgtgtgGTCAATCAATgGTCAATCAATCGTCAATCAATCGTCAATCAATCGTCAATCAATCGTCAATCAATCGTCAAcCAATCGTCAATCAATCGTCAATCAATCGTCAATCAgTCGTCAATCAACCGTCAgTCAATCGTCAATCAATCGTCAATCAATCGTCAATCAATCGTCAATCAATCGTCAATCAATCGTCAATCAATgGTCAATCATCCGCAGCCATTTTGGCTCAAGCCCCCGGCACGTTGGAAGCTCCCTGGCCCCCACCCCCCGCACCGAAGCATG

1234567891011121314151617181920

Intergenic region of Protoceratium luciferasegene consists of 20 repeats of GTCAATCAATC

Lecture 5: 229/26/2006

DNA structure: Primary Structure of DNA: decoding DNA sequences

Lecture 5: 239/26/2006

DNA structure: Primary Structure of DNA: decoding DNA sequences

Dispersed10-401-5IN20-RepAlu tail arrays

Dispersed10-1001-2Apo CII, PLAMicrosatellites

Dispersed10-1003-5FMR-l, MYCN

Short tandemrepeats

Dispersed,subtelomeric10-1009-100Apo B,

D1S7Minisatellites

heterochromatic

Centromeric,≥1000s2-100sAlpha,

BetaSatellites

Genomicdistribution

Array size(repeat units)

Repeat size(base pairs)ExamplesClass

Five classes of simple tandem repetitive sequences

Homework (Wednesday, 9/27/06, Due on Wednesday, 10/2/06)

2. Calculate the exact molecular weights of each of the four standard nucleotides (A, T, G, and C) in DNA molecules?

3. pBR322 is a circular double-stranded plasmid of 4363 nucleotides.Figure out 1) how many picomoles are 1 microgram of the plasmid? 2) How many micrograms are 1 pmol of the plasmid? 3) How much are the weights of two single-stranded circular pBBR322, respectively?

4. Draw diagrams to show all possible base-pairings of adenine and thymine.

1. Show the molecular orbitals of P15.

Lecture 5: 249/26/2006

DNA structure: Primary Structure of DNA: decoding DNA sequences