I don't like the idea that you have to perform gel electrophoresis again n again to check the presence of the gene of interest. is there any alternate method to check it rapidly just like color test i mean.I am just curious about it.
In my experience, running a gel is the best way to directly infer successful transformation. However, you can also try inserting a reporter gene like green fluorescent protein (GFP) downstream of the target gene to indirectly infer transformation. If you sandwich your target gene between your reporter gene and selector gene (antibiotic or rapamycin resistance), there's a good chance that your target gene is being expressed in fluorescent cells growing in selective media. Again, this method does not directly infer expression of the target gene, it just suggests it.
Another method you could try is re-engineering the gene as a GFP fusion protein. Gene expression can be quantified by fluorescent intensity within the cells, and you can see where the protein is being localized within the cells. This is a good method that can directly detect and measure target gene expression, but the drawback is that the fusion protein may not function like the original target protein. Usually, fusion proteins do have the same function as their non-fusion counterparts, but there are exceptions.
If money isn't an problem, you can try running an ELISA on your cell lysate, although now, you'd be running ELISAs all the time instead of gels.
I don't know what gene you're targeting, but if it's an enzyme, there might be a colorimetric assay available as you suggested.
I hope this helps :)
Hi,
if you would like to know if PCR was successful (the presence of PCR product) you can measure increasing DNA concentration (But you have to have Nanodrop or Qubit in post PCR room).
Gel- electrophoresis is the best, you can see non-specific bands before sequencing, the specificity of length of your PCR product and mainly the NEGATIVITY of your negative control!
Yes ther are a ways of detection by color change and absorbance peaks,but detection by PCR is the best
1. If your gene of interest (GOI) is following gene, you can identify transgenic lines by visualizing the phenotype(s) from the transgene products through using microscopes or enzyme reactions, for examples:
- GUS gene: GUS assay
- GFP (RFP, YFP........) gene: can quickly check under GFP microscope
- Luc gene: luciferase assay
- Pigment gene: can quickly identify transgenic lines through color-turning plants
2. Using ImmunoStrips®: usually your GOI is linked to a selectable marker gene (SMG), such as nptII gene (for kanamycin resistance). Assuming the whole cassette (GOI::SMG) is intact in the transgenic lines. Initial screen of SMG can pick out the transgenic lines. For example, NPTII ImmunoStrip® can be used to check whether a transgenic plant has NPTII protein (see attached Figure and Paper). This is probably the best bet for you, because most people use nptII gene as a SMG for kanamycin selection to screen transgenic lines. ImmunoStrip® is pretty easy to use. Just quickly grind up a small tissue and stick the strip into it. If NPTII protein present, one indication band will show up (see attached Figure). Did you use nptII gene as your selection marker?
By the way, for GMO ImmunoStrip® Tests, you can check out this company's product for an example (link below). They also developed ImmunoStrip® to detect Bt gene (ex. Bt corn, cotton) and other common used transgenes. There are probably other companies selling the similar products.
https://orders.agdia.com/gmo-trait-tests/immunostrip-tests
Using PCR is one way to check putative transgenic lines. However, some putative transgenic plants might still have some residual Agrobacterium (not be removed by antibiotics), especially for T0 putative transgenic lines. Therefore, a 'positive' PCR result can be a false positive. False-positive PCR results were observed and reported. Need to be aware of this phenomena.
See two papers:
- "Polymerase chain reaction analysis of transgenic plants contaminated by Agrobacterium" (http://link.springer.com/article/10.1007/BF02772647)
- "An efficient multiplex PCR assay for early detection of Agrobacterium tumefaciens in transgenic plant materials" (see attachment)
In my previous answer, I mentioned pigment genes. Attached is an example from a 2017 publication. The authors studied regulation of anthocyanin pigmentation during growth and development by MYB genes. Overexpression of those genes turn the transgenic tobacco plants from green to purple.
the attached paper describes an isothermal colourimetric method of amplification where the visible colour change indicates increasing amplification so if your primers are good then you just have to look at the tube and the colour of the pH indicator to see presence of an amplimer. The paper also has references to other methods of doing this
may eb u can perform real-time PCR, u can see if the PCR has worked or not and use melt curve analysis.
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