t-dna mutagenesis t-dna mutagenesis. transfer-dna mutagenesis: a chemical or physical treatment that...
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T-DNA Mutagenesis T-DNA Mutagenesis
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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.
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-PURPOSE:
To create loss of function mutations in order to determine the function of a gene.
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-HOW DOES THIS WORK:
Vector transmission by way of Agro plant is randomly inserted into the nuclei chromosomal sites.
T-DNAT-DNA Flanking Region
LBRB
KAN
Pdi2
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-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.
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-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.
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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.
R1 primer
Wild TypePrimer F1
Pdi2 ALB
T-DNA
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-VALID TEST RESULTS:
To achieve valid data from the gel results. Homozygous cells must be used, not Heterozygous.
T-DNA T-DNA Flanking RegionLBRB
KAN
Pdi2
Genotype -
Segregation
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-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.
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-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.
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-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 -
WT A1 A2 WT+ - WT A1 A2 TDNA+ -
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WT A1 A2 WT+ - WT A1 A2 TDNA+ -
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WT A1 A2 TDNA+ - WT A1 A2 WT+ -
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-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.
R1 primer
Wild TypePrimer F1
Pdi2 ALB
T-DNA
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Future Advances in Biotechnology Mutagenesis Research:
BEFORE
AFTER