I'm trying to find the transgene integration sites (with sequences) of a transgenic variety of Maize please suggest me a advanced and good method for this, this will help me to do my research more fast & advanced?
If you want to find out the precise location of insertion, and if you are using T-DNA transformation. You should find out the adjacent sequence (the plant genomic sequence) next to the T-DNA. A few methods were published, and one of them is to use iPCR (inverse PCR).
http://www.ncbi.nlm.nih.gov/pubmed/22065444
"Identification of DNA sequences that flank a known region by inverse PCR".
The other way is using thermal asymmetric interlaced PCR (TAIL-PCR).
http://www.sciencedirect.com/science/article/pii/S0168945203002188
"Obtaining and analysis of flanking sequences from T-DNA transformants of Arabidopsis"
In addition to Yuan-Yeu's answer, and if your transgenic is a T-DNA integration, I would suggest you find out the insertion copy number first. If you expect a low copy number (1-2) qPCR with genomic template can be reliable, otherwise Southern Blot should be the method of choice.
Once you know the copy number, iPCR, TAIL and other genome walking methods can be used. The problem you face if you don't know the copy number is that those methods are not robust, so if you can only detect the flanking sites for two, but in reality you have three insertions and the third happens to be the reason for your key phenotype (e.g. by physically altering the expression of a gene), you will miss it.
If you have a good budget and are pressed by time, I would suggest using a genome walking kit. The are usually more robust that iPCR and TAIL and block spurious products more efficiently.
Best,
Ruben
Ruben raised a good point. It will be good if you know the transgene copy number first. Multiple insertions might affect the efficiency of using iPCR or TAIL PCR. In addition, multiple transgene insertions can cause gene silencing. So, it will be good you can conduct Southern assays first. Besides, detailed Southern analysis can also tell you whether or not your transgene is intact or truncated. If truncated, where is the roughly truncated point. With those information, you can further design your experiments using methods mentioned above to look for the exact adjacent sequence to the T-DNA.
I also found this 2013 paper. They mainly used targeted genome sequencing to find out the insertion sites. They also pointed out the advantages of using this method over TAIL PCR or IPCR (yellow highlights). See attached paper. It should be useful to you.
"Time- and Cost-Efficient Identification of T-DNA Insertion Sites through Targeted Genomic Sequencing"
The transgenic plant I'm working on is transformed with Particle Bombardment, so which method will be relevant for this?
By using TLA (targeted locus amplification).
See more info here: http://www.cergentis.com/cergentis/about
It is better and gives more choice when you have plenty of independent events to choose interesting ones from. If you have many independent events (you can estimate the plants arising from different callus) you can evaluate them phenotypically and later carry the analysis forward to sequence level.
At first, you can screen for low copy number lines by qPCR (https://pubmed.ncbi.nlm.nih.gov/15459795/).
Having low copy number lines, you can use 'gene walking' techniques (inverse PCR, TAIL PCR) to estimate the point of insertion (https://pubmed.ncbi.nlm.nih.gov/22065444/; https://www.sciencedirect.com/science/article/pii/S0168945203002188).
With available time and resources, you can rely on NGS - Targeted locus amplification (TLA) (https://pubmed.ncbi.nlm.nih.gov/27822865/#:~:text=Targeted%20locus%20amplification%20(TLA)%20is,a%20short%20locus%2Dspecific%20sequence.)
All the best ☺️
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