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Q26 - T-DNA Southern Blot Analysis

Theoretical 2 Real exam question - full text reproduced under IBO's CC BY-NC-SA 4.0 license
Recombinant DNA & Biotechnology Techniques Biomolecular Analytical Techniques

During Agrobacterium-mediated transformation of plants, one or more copies of T-DNA (Transfer DNA) can integrate randomly into the plant genomic DNA.

The number of T-DNA insertions can be analyzed by Southern hybridization of digested genomic DNA from transformed plants, using probes against sequences within the T-DNA. The restriction enzyme used for digestion of genomic DNA should preferably have a single cut site within the T-DNA. The restriction enzyme would cut within the integrated T-DNA, and also in the flanking genomic DNA.

Figure 1: Vector (15 Kb) used for plant transformation. The T-DNA region (5 Kb) lies between the LB (left border) and RB (right border). Rectangles A and B represent probe regions. X and Y denote restriction sites.

Figure 2: Southern hybridization results for DNA from independently transformed plants (1 to 5) and untransformed plant (UT), with probe A and probe B.

True or False?

Q26.1. The genomic DNA was digested with restriction enzyme Y.
Q26.2. The digested DNA fragments that hybridized to the probe contain regions of T-DNA only.
Q26.3. There are two copies of T-DNA in plant number 1.
Q26.4. One copy of T-DNA is integrated in plant number 2.
Q26.5. Complete T-DNA region has been transferred in plant 4.
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Question reproduced from IBO 2023, Theoretical Paper 2, licensed under CC BY-NC-SA 4.0 - attributed to the International Biology Olympiad. Open the full exam PDF