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Solar UV Radiation. UVC <290 nm Absorbed by ozone UVB 290 – 320 nm ~0.5 % total energy UVA 320 – 400 nm 6% total energy. UV Spectrum of DNA. Absorbance. 300. 250. Wavelength (nm). T<>T. T<>C. C<>C. Pyrimidine Photodimers. Bipyrimidine (6-4) Photoadducts.

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Presentation Transcript
solar uv radiation
Solar UV Radiation
  • UVC <290 nm Absorbed by ozone
  • UVB 290 – 320 nm ~0.5 % total energy
  • UVA 320 – 400 nm 6% total energy
uv spectrum of dna
UV Spectrum of DNA

Absorbance

300

250

Wavelength (nm)

dna photooxidation mechanisms
DNA Photooxidation Mechanisms

Oxidation at

GG(A) doublet

P

G Oxidation

Hole Transfer

DNA

Type I

Type II

P-.+ DNA+.

P*

1O2 singlet

oxygen

O2

DNA

method of irradiation and visualization of dna damage
Method of irradiation and visualization of DNA damage

UV irradiation

32P-DNA + PS

Upturned Eppendorf lid

EtOH precipitation

 Piperidine

Electrophoresis

Autoradiography

single stranded dna
Single-stranded DNA
  • Singlet oxygen
  • UV dose & [SP] dependent

Uniform

G-specific attack

quenching in ss dna
Quenching in ssDNA

1SP + UVB

2 & 3Quenched by sodium azide & DABCO

4 & 5Mannitol & SOD have no effect

duplex dna
Duplex DNA

Specificity for 5’-G of 5’-GG-3’ doublets

5’-G of 5’-GA-3’ doublets

conclusions
Conclusions
  • Single-stranded DNA - Type II mechanism
    • singlet oxygen
  • Duplex DNA - Type I mechanism
    • electron transfer - DNA to excited sensitizer
    • hole migration
maldi tof analysis of dna
MALDI-TOF Analysis of DNA
  • Requires picomole amounts of sample
  • Mass accuracy to ~0.1%
  • Generate +ve and –ve ion spectra
  • UV-absorbing matrix for <100 base pairs
  • Applications in DNA sequencing
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