Solar uv radiation
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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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Solar UV Radiation

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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)


Pyrimidine photodimers

T<>T

T<>C

C<>C

Pyrimidine Photodimers


Solar uv radiation

Bipyrimidine (6-4) Photoadducts


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


Singlet oxygen damage in ss dna increases in d 2 o

H2O

D2O

Singlet oxygen damage in ss DNA increases in D2O


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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