Transcript The elabration of RAMD-PCR assay for detection of a

PCR assay of intragenic mutation lesions
induced by monoenergetic fission neutrons
and gamma rays in Drosophila
Part I: Gamma rays
Dr Igor Donatovich Alexandrov
Genetic Group
Laboratory of Nuclear Problems
Nanette Brand1
Nonhlanhla Ngwenya2
1Stellenbosch University, 2University of Pretoria, South Africa
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
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
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
Black gene structure
A
5’
DNA
Ex1
Ex2
In 1
Ex3
DNA
3’
In 2
’
B
F1
R1
F3
R3
Fragment 1
Fragment 3
F2
R2
Fragment 2
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
Primer sequence for PCR
Fragment
1
2
3
Primer
Primer sequence
F1
aggtgagatcggcacctg
R1
ttggctgcaatggggcactcac
F2
acaacactcgcccgagtcca
R2
acactgttgcaggcagc
F3
tggttgctcatttcgaggggt
R3
tcccagttcccaaggcaaggac
Annealing
temperature
(○С)
Size of
the PCR
product
(bp)
64
1068
Size of the
overlap
fragment (bp)
45
64
1043
99
64
859
Methods

DNA isolation

PCR assay

Gel electrophoresis (DNA analysis)
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

Electrophoresis
1
2
3
4
5
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

Acknowledgements
Dr I. Alexandrov, Dr M. Alexandrova and
Liliana Namolovan
 Co-presenter

Thank You for Your attention
Protocol for DNA Isolation
Homogenization of
tissue
Binding of DNA
with sorbent
Purification step
Purified DNA
1
2
3
4
5
6
7
8
9
10
Lane 1-3 = 1st fragment, Lane 4, 5 & 7 = 2nd fragment, Lane 8-10 =
3rd fragment and Lane 6 = DNA marker