Hi All
I am planning to do southern blotting in order to detect the transgene copy number in plants. Can anyone please suggest me how can I design my prob? Also, which method of detection, radioactive/DIG will be good for this purpose?
Hii Hassan..you can label your probe using PCR product or restrictio digestion. Both methods of detection works. I use DİG labelled proble and it does not disappoints me.
If you want to avoid radioactivity you can use biotinylated dNTPs. You can purchase them from any supplier you wish and then use an anti-biotin antibody (I used hrp conjugated to detect with ECL).
For the probe design and choice of restriction enzyme(s) it is important that you are able to detect one of the borders of your T-DNA along with the flanking plant DNA. The flanking plant DNA in different transgenic lines and for multiple inserts in the same line will vary, so this will enable you to determine the copy number.
The DIG system works very good for plant DNA, in which you can label your probe using PCR. The probe length can be several 100bp up to 3kb. Mostly you obtain so much product from PCR that you can make several hyb mixes from that, also you can reuse all solutions including the hyb mix several times. Ideal when you need to make a lot of blots for the same probe.
I suggest you copy number detection in transgene, radioactive method is one of the best method and use, probe around 500bp in the clone digestion product because the PCR product gives randomly binds
I also suggest the radioactive method.you can take the amplified product of the gene from the plasmid and label it using labelling kit .Alternatively you can do the restriction digestion of the plasmid and the released insert can be used for labelling.PCR method is comparatively easy ,if the correct primers and the amplifying conditions are available.
I would suggest performing a real-time quantitative PCR rather a Southern. Southern blotting is quite laborious and not always easy to interpret.
I would clone, in the same plasmid, one fragment of the transgene and one fragment of a single copy endogenous gene. You may have a look on the use of plasmid calibrants for the quantification of transgenic plants ( Eugénia De Andrade Silva, B. Jeynov, P. Corbisier, A. M. Kortekaas, S. Trapmann, S. Vincent, H. Emons Certification of a Maize NK603 Reference Material for its DNA Copy Number Ratio. Certified Reference Material ERM®-BF415e. Certification Report EUR 24699 EN; doi:10.2787/38148). I would advice to clone only parts of the transgene without any plant junction DNA (5’ or 3’ junctions) because each integration site is unique and you want to assess all integration-sites. Additionally, you have to clone different parts of the transgene because it could happen DNA rearrangements and only some parts of transgene could integrate somewhere else. In this way you determine the number of insertions and also possible rearrangements. The TOPO XL plasmid allow for long inserts, up to 20 Kb.
You may also try chromosome in situ hybridization. I do not know which plant species you are dealing with. It can be that the chromosomes are too small for FISH detection. It only a suggestion.
I did radioisotope labelling for southern blotting. Simply, design probe specific PCR primers and go for PCR amplification. Use hexalabel kit for labelling the probe and use in southern blotting.
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