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Nanette Brand 1 Nonhlanhla Ngwenya 2

PCR assay of intragenic mutation lesions induced by monoenergetic fission neutrons and gamma rays in Drosophila Part I: Gamma rays. Nanette Brand 1 Nonhlanhla Ngwenya 2 1 Stellenbosch University, 2 University of Pretoria, South Africa. Dr Igor Donatovich Alexandrov Genetic Group

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Nanette Brand 1 Nonhlanhla Ngwenya 2

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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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