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PCR assay of intragenic mutation lesions induced by monoenergetic fission neutrons and gamma rays in

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1Stellenbosch University, 2University of Pretoria, South Africa. Dr Igor Donatovich ... Physical map of black gene showing introns (In 1-2) and exons (Ex 1-3) ... – PowerPoint PPT presentation

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Title: PCR assay of intragenic mutation lesions induced by monoenergetic fission neutrons and gamma rays in


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

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
DNA Ex1
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
7
6
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
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14
Protocol for DNA Isolation
Homogenization of tissue
Binding of DNA with sorbent
Purification step
Purified DNA
15
1
2
3
5
4
7
6
8
9
10
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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