Download T-DNA Mapping

ABE workshop 2007
T-DNA mapping by PCR
Eun Ju Cho
Objectives
To more fully understand the function of PDIs in plants, we are
using a method called “gene knockouts”, which allows the
determination of the resulting effects of the absence of a specific
gene on growth, development, cell physiology and biochemistry.
The gene is knocked out by disruption with a DNA insertion
that contains a kanamycin resistance gene (kanR), called a “TDNA”.
What is the T-DNA?
“Transfer”-DNA (T-DNA) can be randomly inserted into the
plant genome by Agrobacterium tumefaciens. The T-DNA
contains a selectable marker (kanamycin resistance) and
other genes for growth of the Agrobacterium. The insertion
of T-DNA into a gene disrupts the gene (“knockout”) and the
function of the protein it encodes.
Genetic Map of T-DNA knockout mutant of PDI2
LB1
~200bp
T-DNA
UTR
LP
~300bp
Exon 1
E2
UTR
RP
ATG
890bp
At1g52260(PDI 3)
Chromosome 1
LP - Left genomic primer (CCAAAATTAAAAACCAAAAAGCAA)
RP - Right genomic primer (TCAAGAAATCTCGGAGCTTCA)
LB1 - Left border primer of the T-DNA insertion:
( GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC)
T-DNA sequence of PDI3
SAIL_302_G10(1071bp)
aaatcgaattaattcggcgtaattacgacattaaaaacgtccgcaaggtgttatataagt
tgtctaagcgtcaatttgtgataggaattcgaagattgcatcggagcttgagatataaag
gtttccctacgcttcttctctactgctcctacacaannnnnnggagccannntccgacgg
acggaacagaggcaccagaggccaacacactgaaaagatcattggctgaatacacaaccg
aaggtgaagagagagccacaaggaaaacacaaaaacccccaggactaagcgcccgctcaa
tccactccaaaaaacacgaccgaataaagcgcactgataaaacacactgaacaacccaaa
accacgaagttgcatcatcgaacggctggcagagaaaaagaaccattagacaaacaacgc
gcggacatggaacccagacacatacccaacgagaacgcatacgcctacaaatcgcggggt
cccaacaaaaaacagcacccgcgccccacaccacacaccgcagtgaagagaacaacacga
aacaagaaaaccccgaagagagcgccccccaccccgcacgtgaggaccagccgagacgaa
ccagggcggggcacagagagaacacaagggggaacgagagggggcacgcacagagcagga
gccaaacgggccagcccaaccccaccaccgcccccacacaacgacgcccaccccacccga
cgacccacccccaccgcgcccaacccaccgcaaccccaccccccccgccacccccactcc
ccgcggcgcccaccaccacgccccacgcccacacgccaccgcacgacccaanaacacccc
gcccgccccacccgccacgaaccgcacccaccgcgccccccaccaccaccccccgccccc
cccgccgacgcccgccaccaccacacacgcaccccccaaacccccccaccaccaccccac
gcccgcgcgcacccccccccnncccanaacccgccgccccccccccccaacacccgcccc
cgccacccccgcgcccgcccgccgcnaccccccacccccaccccaccaccn
PCR mapping for T-DNA knockout mutant of PDI 3
M
HM
HZ
WT
1kbp
T- DNA Primer design
LP + RP + LB1
WT : no insertion (890bp)
HM : insertion both chromosome (~500bp)
HZ : one of the pair chromosome with insertion
517bp
Condition
94℃
5min
94 ℃
30sec
55 ℃
30sec
72 ℃
1min
72 ℃
10min
30 cycle