Detection of DNA by Fluorescence Method

1
Detection of DNA by Fluorescence Method
2
Methods of DNA Detection

• Radioactive Labeled Probes.
• Silver Stain Method.
• Fluorescence Detection.

3
Introduction

• Chemiluminescence is the generation of
  electromagnetic radiation as light by the release
  of energy from a chemical reaction.
• You do not need to give any energy to get light.

4
Introduction

• Bioluminescence is simply light produced by a
  chemical reaction which originates in an
  organism.
• e.g. Firefly, Jellyfish

5
Introduction

• In fluorescence you need to give the energy
  (either laser or UV) to get the light.
• It has the short life time.
• e.g. Ru(bpy)32

6
Introduction

• Phosphorescence is similar to fluorescence.
• It has long life time.
• e.g. Compounds in Clocks and Watches dial.

7
Fluorescence

• A fluorophore, in analogy to a chromophore, is a
  component of a molecule which causes a molecule
  to be fluorescent.
• It is a functional group in a molecule which will
  absorb energy of a specific wavelength and
  re-emit energy at a different (but equally
  specific) wavelength.
• e.g. Polyaromatic Hydrocarbon or Heterocycles

8
Fluorescence
Fluorescence of Few Minerals
9
Fluorescence

• Fluorescence intensity is quantitatively
  dependent on the same parameters as absorbance.
• Log (Io / I) ?cd A
• (Lambert Beer Law)

Unabsorbed Light
Incident Light (UV or Laser)
Detector
10
Fluorescence

• DYES THAT FLUORESCE IN VISIBLE SPECTRUM.
• EXCITED BY LASER or UV.
• GROUND STATE gt EXCITED STATE gt
  CONFORMATIONAL CHANGE gt PHOTON EMITTED.

11
Fluorescence
Normalized Fluorescent Intensity
560
620
Wavelength ?
Emission or Luminescence Spectrum
12
Fluorescence

• The process responsible for the fluorescence
  properties of fluorescent probes and other
  fluorophores is illustrated by the simple
  electronic-state diagram called a Jablonski
  diagram.
• Polish Scientist called Professor Alexander
  Jablonski proposed in 1935

13
Fluorescence
14
Fluorescence

• When an organic molecule absorbs energy and gets
  promoted to an excited state.
• The molecule in the excited state reaches the
  ground state by three different way.
• 1. Photochemical Reaction
• 2. Radiationless or Non Radiative Decay
• 3. Fluorescence.

15
Fluorescence

• If a molecule contains two light absorbing
  species, able to rotate with respect to one
  another, it increases the probability of
  Radiationless or Non Radiative process
  (Emission of Heat).
• If something restricts the rotation, the molecule
  start to fluorescence (Emission of Light).

16
Fluorescence

• Our molecule has two light absorbing unit.
• Rotation between two light absorbing unit is not
  possible due to CH group.

D 7
17
Procedure

• Dissolve a small amount of crystallized D7 dye
  in 5 mL of hot water.
• Cool to room temperature and wait until the
  excess of compound settled down.
• Take 5 drop from the above solution (D7) and
  dilute with 5 mL of water.
• Split into two parts in a small test tube.

18
Fluorescence

• Add three drops of the Calf Thymus DNA into one
  of these test tubes and swirl the mixture.
• Keep your solution in a dark place and irradiate
  UV light on both test tube.
• You will see a green fluorescence in the test
  tube which contains DNA.

19
Notes

• Work with the same group.
• Give me back the unused D7 dye.
• After your experiment, I will take you down to
  explain how the spectroflurometer work.

20
Note

• Your Lab note book graded
• 1. Organization Format 40 Pts
• 2. Table of Contents 40 Pts
• 3. Pages Numbered and Dated 40 Pts
• 4. Data Tables 40 Pts
• 5. Correction and Neatness 40 Pts
• 0 for no information if it is half written then
  we gave 20 and 40 for good work.