I am trying to locate a putative insertion of almost 8.5 kb in a Drosophila mutant by Long Range PCR using Platinum High Fidelity Taq Polymerase. The insertion does not appear in every PCR, although I use the same genomic DNA. Can someone help me how to confirm its presence?
Have you integratet a complete plasmid into the genome or just a gene with 8.5 kb lenght??
If you please, I should like to offer a creative suggestion.
Construct a primer that has the property of being a shustring of the genome in question, and particularly a shustring that occurs within the body of your target, such as toward the 5' end of your target. If your liquor contains only sample DNA (no contaminants), then the primer will be able to interact only with your target (your hoped-for insertion). If you get amplification, then your insert was successful.
if you know the site of insertion and the sequence of the part you inserted in the genome ..... just design primers from extream ends of inserted fragments and from the flanking regions of site of insertion and go for PCR in different combination it will be easy to amplify the shorter length and it will also confirm you the direction of insertion.........
Besides PCR you might give hybridization-based techniques a try. With PCR, you might suffer otherwise from two of the core problem(s) of this technique for the purpose of detecting insertion: over-optimization and trace contaminations. If you try to optimize the PCR until you get a product, you might end up with a band of approximately the same size as the desired product but which is a result of wrong priming. Can be solved be sequencing the product, of course. But if you have a trace contamination, even that would not help.
So if you know at least a part of the sequnce of your insert, you might try Southern blotting and hybridization or maybe even fluorescence in situ hybridization. More work of course but much more reliable of done correctly.
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