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

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 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

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.

Effects of radiation • irradiation: γ 40 Gy • damage of DNA  mutations Point mutations Substitution Insertion Deletion

Drosophila melanogaster as a model organism

Phenotypes of mutations Picture 1. Wild phenotype of Drosophila melanogaster Picture 2. Phenotypes of vestigial, black, cinnabar, yellow, white All of the studied flies are from next filial generations of irradiated subjects.

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 of yellow gene amplicons under study.

Work sequence • standard DNA isolation protocol 1 • polymerase chain reaction 2 • electrophoresis 3 • evaluation and discussion of results 4

DNA isolation Homogenisation • 10 – 20 flies per 1 sample were processed and turned into DNA samples (16 samples in total) Incubation centrifugation Absorption on NucleoS TM Mixing washing Releasing with Extra Gene TM vortexing DNA

PCR Diluent + water Adding primers 94 ° C, 1 min / one cycle Adding DNA 32 – 65 ° C, 0.25 sec - 1 min 25 – 40 cycles 72 ° C, 1 min Mineral oil one cycle PCR

Gel Electrophoresis • separation of DNA fragments on agarose gel (1%) in buffer: Tris-boric acid-EDTA + ethidium bromide ( visualizing dye) • sampling: ~18 μ l • conditions: 100 V, 20 min. • visualisation: UV

Electrophoresis

Results – visualised gels

Discussion

- 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 .