I have generated several transgenic Arabidopsis lines by Agrobacterium mediated transformation. Now, I want to map the genomic position of T-DNA in these lines. What will be the best and easiest way of finding genomic insertion sites of the transgene in these lines?
First check where is the ends of your T-DNA. Some T-DNA will be intact, and some T-DNA will be truncated. Once you identify the end of your T-DNA, you can design primers for Inverse PCR or other means to fish out the junction sequence of the host genomic DNA. After you get the info of the host genomic DNA (joint to the T-DNA end) sequence, you can BLAST this sequence to this particular plant genomic sequences in the database (if the plant genomic sequence is known) and figure out where is the insertion location at.
See publication by Lepage et al. in PLOS One
Time- and Cost-Efficient Identification of T-DNA Insertion Sites through Targeted Genomic Sequencing
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070912
Yes, if TAIL PCR proves difficult you should try inverse PCR followed by sequencing. To determine the number of T-DNA integrants you can try a genomic southern with different restriction enzymes that do not cleave within the T-DNA. The number of unique bands and their intensities on the southern should tell you the number of integrants within the genome. This should match the answer you get by inverse PCR, unless of course as stated by Yuan, some of the T-DNA integrants are partial and you are missing the primer binding sites for the inverse PCR.
I suggest the use of TAIL-PCR to know exactly where your T-DNA is inserted in the plant genome.
Regards
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