induced by ionizing radiation at the paratelomeric yellow gene of - - PowerPoint PPT Presentation

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induced by ionizing radiation at the paratelomeric yellow gene of - - PowerPoint PPT Presentation

Dec 03, 2022 27 likes •260 views

PCR-assay of the mutational lesions induced by ionizing radiation at the paratelomeric yellow gene of Drosophila melanogaster Team work Radio genetics group Dzhelepov Laboratory of Nuclear Problems Joint Institute For Nuclear Research

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PCR-assay of the mutational lesions induced by ionizing radiation at the paratelomeric yellow gene of Drosophila melanogaster

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Team work

Overall Leader:

Igor D. Alexandrov

Ph.D. Dr. Sci. (Biology), chief, sci. res.

Practical approach Leader:

Kristina P. Afanasyeva

Ph.D., sci. res

Members:

Diana Raei, Sarah Awam, Seham ElMarakby, Mohamad Taha

Radio genetics group Dzhelepov Laboratory of Nuclear Problems Joint Institute For Nuclear Research

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Aim of the work

  • The goal of the Project is to detect the nature

and location of DNA alterations induced by γ- rays at the paratelomric yellow gene of Drosophila melanogaster.

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Effects of radiation

  • irradiation: γ 40 Gy
  • damage of DNA  mutations

Substitution Insertion Deletion

Point mutations

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Drosophila melanogaster as a model

  • rganism
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Phenotypes of mutations

Picture 1. Wild phenotype of Drosophila melanogaster

All of the studied flies are from next filial generations of irradiated subjects.

Picture 2. Phenotypes of vestigial, black, cinnabar, yellow, white

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Picture 3. a) scheme of the first politen chromosome; b) genetic map of yellow gene region; c) yellow gene exon-intron structure (4737 bp); d) location

  • f yellow gene amplicons under study.
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Work sequence

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  • standard DNA isolation protocol

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  • polymerase chain reaction

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  • electrophoresis

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  • evaluation and discussion of results
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DNA isolation

  • 10 – 20 flies per 1 sample were processed and

turned into DNA samples (16 samples in total)

Homogenisation Incubation Absorption on NucleoSTM Releasing with Extra GeneTM DNA

Mixing washing centrifugation vortexing

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PCR Diluent + water Adding primers Adding DNA Mineral oil PCR

32 – 65°C, 0.25 sec - 1 min 25 – 40 cycles 72°C, 1 min

  • ne cycle

94°C, 1 min / one cycle

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Gel Electrophoresis

  • separation of DNA fragments on agarose gel (1%)

in buffer: Tris-boric acid-EDTA + ethidium bromide ( visualizing dye)

  • sampling: ~18 μl
  • conditions: 100 V, 20 min.
  • visualisation: UV
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Electrophoresis

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Results – visualised gels

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Discussion

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  • Among 12 mutants , Only 6 have chromosomal aberration which

all were investigated by PCR. Four of these 6 mutants have no detectable changes by PCR which conclude that these changes had happened outside our gene of interest and but they still have a phenotype change which means the need to be investigated with another method rather than PCR.

  • 2 of the 6 mutants with chromosomal aberration have missing

fragments of yellow gene which conclude that Gamma radiation caused genetic loss inside the yellow gene.

  • 6 mutants have no chromosomal aberration , however PCR could

detect genetic changes in one of them.

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