Title: PCR assay of intragenic mutation lesions induced by monoenergetic fission neutrons and gamma rays in
1PCR 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
2Goal
- 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
3Polymerase 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
4Model 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
5Black 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
6Primer sequence for PCR
7Methods
- DNA isolation
- PCR assay
- Gel electrophoresis (DNA analysis)
8Results
- 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
9Electrophoresis
2
3
5
10
1
4
7
6
8
9
10Conclusion
- 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
11Acknowledgements
- Dr I. Alexandrov, Dr M. Alexandrova and Liliana
Namolovan - Co-presenter
12- Thank You for Your attention
13(No Transcript)
14Protocol for DNA Isolation
Homogenization of tissue
Binding of DNA with sorbent
Purification step
Purified DNA
151
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