Download T-DNA Mutagenesis

T-DNA Mutagenesis
Transfer-DNA
Mutagenesis:
a chemical or physical treatment
that creates changes in DNA
sequence which can lead to
mutation strains that are passed
on to the next generation.
-PURPOSE:
To create loss of function mutations in order to determine the function of a
gene.
-HOW DOES THIS WORK:
Vector transmission by way of Agro plant is randomly inserted into the
nuclei chromosomal sites.
RB
T-DNA
LB
T-DNA Flanking Region
KAN
Pdi2
-Agro?
Agrobacteria tumefaciens is a bacteria found on certain plants that were
found to cause tumors on wounded plant areas. Found to contain Ti
(Tumor inducing) plasmid that creates a mutation in the plants genomic
sequence. The Ti plasmid’s ability to integrate itself into a DNA sequence
was isolated and the tumor inducing quality was taken out.
-LOSS OF FUNCTION MUTATIONS:
is when a mutation is created in such a way that death does not occur so as to
observe the effects on the plant by the loss of a certain gene. In other words, a
gene is knocked out and the plant is grown and observed for any differences
between the mutant strain and the control strain. Thus facilitating
(understanding) the function of that knocked-out gene.
T-DNA in the Arabidopsis Genome
T-DNA –
from Ti plasmid in the Agrobacteria tumefaciens..
Uses the insertional quality to carry foreign genes into the plant genome.
Pdi2 A
LB
T-DNA
Primer F1
Wild Type
R1 primer
-VALID TEST RESULTS:
To achieve valid data from the gel results. Homozygous cells must be used, not
Heterozygous.
RB
T-DNA
LB
T-DNA Flanking Region
KAN
Pdi2
Genotype -
Segregation
-HOMOZYGOUS:
2 of the same (genes). On gel, homozygous will only produce one band on
either the wild type control side or the T-DNA control side, not both. Needed for
accurate results.
-HETEROZYGOUS:
different genes. On gel, will produce a band on both the WT control side and
the T-DNA control side. Not used for verification of T-DNA.
-GEL ELECTROPHORESIS:
1. Verify if sample is homozygous, 2. Verify that T-DNA knockout is not there, 3.
Verify that T-DNA is there and inserted.
WT A1
A2 TDNA+ - WT+ A1 A2
WT A1
A2
WT+ -
WT
-
WT
A1 A2 TDNA+ -
WT
A1
A2
WT+
-
WT
A1
A2
TDNA+
-
WT
A1
A2
TDNA+
- WT
A1
A2
WT+ -
-disrupted genes do not make RNA. That function has been knocked out.
-F1 and R2 primer will make WT.
-F1 and LB primer will make T-DNA knockout.
Pdi2 A
LB
T-DNA
Primer F1
Wild Type
R1 primer
Future Advances in Biotechnology Mutagenesis Research:
BEFORE
AFTER