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Genetic Effects of Fission Neutrons & Gamma Rays in Drosophila: PCR Assay Study

Study on gene mutations in Drosophila induced by fission neutrons and gamma rays, using PCR assay to detect DNA alterations. Comparison of mutational lesions. Molecular genetic action of gamma rays on black mutant gene examined.

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Genetic Effects of Fission Neutrons & Gamma Rays in Drosophila: PCR Assay Study

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  1. PCR assay of intragenic mutation lesions induced by monoenergetic fission neutrons and gamma rays in DrosophilaPart I: Gamma rays Nanette Brand1 Nonhlanhla Ngwenya2 1Stellenbosch University, 2University of Pretoria, South Africa Dr Igor Donatovich Alexandrov Genetic Group Laboratory of Nuclear Problems

  2. Goal • To detect the quality and frequency of neutron-induced mutational lesions in comparison to gamma ray-induced ones for different genes of Drosophila using PCR assay • Our aim: To study the molecular genetic action of gamma rays (60Co) on the black mutant of Drosophila

  3. Polymerase Chain Reaction • The polymerase chain reaction (PCR) is a technique for the in vitro amplification of specific sequences of DNA • PCR allows the detection of different kinds of mutational changes within fragments, • deletions • locations • PCR result can be positive or negative

  4. Model of study A B Drosophila melanogaster (A) Wild type, (B) Black mutant • Well studied example, gene structure known • Has common principal DNA structure with humans • Short life cycle (~15 days) • Permits the study of heritable gene mutation

  5. Black gene structure DNAEx1 Ex2 Ex3 DNA A 5’ 3’ In 1 In 2 ’ F1 R1 F3 R3 Fragment 1 Fragment 3 F2 R2 Fragment 2 B A. Physical map of black gene showing introns (In 1-2) and exons (Ex 1-3). B. Sizes and location of the black gene fragments studied with forward (F) and reverse (R) primers

  6. Primer sequence for PCR

  7. Methods • DNA isolation • PCR assay • Gel electrophoresis (DNA analysis)

  8. Results • 22 black mutants studied • 66 PCR assays performed • Deletion of 2 fragments for 1 black mutant was detected • 21 black mutants have a small DNA alterations not detected by PCR

  9. Electrophoresis 2 3 5 10 1 4 6 7 8 9

  10. Conclusion • Gamma rays induce mostly small DNA alterations which cannot be detected by PCR • This study serves as a basis for a study of the molecular genetic action of neutrons

  11. Acknowledgements • Dr I. Alexandrov, Dr M. Alexandrova and Liliana Namolovan • Co-presenter

  12. Thank You for Your attention

  13. Protocol for DNA Isolation Homogenization of tissue Binding of DNA with sorbent Purification step Purified DNA

  14. 2 3 5 7 10 1 4 6 8 9 Lane 1-3 = 1st fragment, Lane 4, 5 & 7 = 2nd fragment, Lane 8-10 = 3rd fragment and Lane 6 = DNA marker

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