I did touchdown PCR using gene specific primer targeting lectin gene inserted into the cotton genome by isolating DNA from T2 plants.The PCR showed positive result in one of the event plant. The seeds from the positive T2 plant obtained from selfing were sown again in T3 and then PCR was done with the same program by using the DNA isolated from T3 plants( sown plants) as template. But non of them is showing positive for the same touchdown program with the same gene specific primer. Is it ever possible and that to in T3 generation?
Dear Sahu, many factors can lead to the results you have obtained, some of them are as follows
1. Do the transgene integration in T2 plants confirmed by robust methods other than PCR, such as Southern, Northern or Western, use of touchdown PCR indicates difficulty in PCR amplification, it is important to confirm the transgene integration in T2 plants
2. If the selected T2 plant is hemizygous for the transgene, then it would segregate in 3:1 ratio, (single copy), since you have not mentioned how many seedlings were used for screening, in case you have used few seedlings, it may help to screen more progeny, we routinely use 45-60 progeny.
3. Primer binding site rearrangement would be unlikely, but could lead to such results. Try redesigning primers targeting other T-DNA regions.
4. Problems with PCR/gDNA. In case, there is a time gap while performing PCR in T2 and T3. Failure of any of the PCR components in T3, including primer degradation or repeated freeze/thaw might lead to PCR failure. Use some positive controls,try amplifying some known single copy native gene/s employing same PCR master mix.
Good luck
You can also try other regions of T-DNA
Please always use positive and negative control while screening putative transgenic plants. You can desigh a pair of different gene primers and screen all your T3 plants. However in case of transgennic you should get positive plants in T3 generation IN PCR screening, if these are derived from positive transgenic T2 plant.
Hi Anirudth, I want to add in this discussion...how many seeds u have collected from T2 positive plant is one of the important thing,.......also have you checked its homozygosity ( southern hybrdization)...this will decide whether it will segregate in T3 or not..
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