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.

T-DNAT-DNA Flanking Region

LBRB

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.

R1 primer

Wild TypePrimer F1

Pdi2 ALB

T-DNA

-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

-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 -

WT A1 A2 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.

R1 primer

Wild TypePrimer F1

Pdi2 ALB

T-DNA

Future Advances in Biotechnology Mutagenesis Research:

BEFORE

AFTER