Given below is a schematic representation of the T-DNA region of a binary vector used for genetic transformation of plants. The figure shows the presence of restriction sites for EcoRV and probes (labelled 1- 4) for Southern hybridization to analyse copy number of T-DNA in the transgenic plants.

Given below are probes or combination of probes that were used by researchers for Southern blotting following digestion of genomic DNA with EcoRV.

A. Probe 3 and Probe 4
B. Probe 1 and Probe 3
C. Probe 1 only
D. Probe 2 only
E. Probe 1 and Probe 4

Which one of the following options represents the correct combinations of probes that would identify single copy integration events from BOTH flanks of T-DNA?"

Correct option is C

The correct answer is: (3) A, D and E only

Explanation : To detect single copy integration events from both flanks of T-DNA, we need to combine probes that specifically target the left and right border regions of the T-DNA. Based on the provided diagram, probes that can hybridize on both flanks (left and right) of T-DNA will effectively identify single copy integration events.

• Probe 1 hybridizes to the left flank (position 1).

• Probe 4 hybridizes to the right flank (position 4).

• Probe 3 targets the gene (which may span across the left flank, providing additional information on integration).

• Probe 2 focuses on the right flank.

To accurately identify integration events from both flanks, the combinations of probes must cover both the left and right borders of T-DNA.

• Probe 1 (left flank) and Probe 4 (right flank) together help identify integration events across the entire T-DNA region.

• Probe 3 (gene) and Probe 4 (right flank) offer a suitable combination to ensure both flanks are detected during hybridization, targeting regions both inside and around the T-DNA border.

Thus, combining Probe 1, Probe 4, and Probe 3 provides the best coverage for identifying integration events from both T-DNA flanks, making option (3) A, D, and E the correct answer.

Information Booster:

1. Probe 1 hybridizes to the left flank of T-DNA (position 1).

2. Probe 3 targets the gene region that spans both flanks and helps verify integration.

3. Probe 4 hybridizes to the right flank (position 4), which helps detect integration on the right side.

4. These probes combined can ensure the detection of T-DNA integration from both left and right flanks.

5. Southern hybridization is used to analyze the integration and copy number of the T-DNA.

6. Identifying integration events across both flanks is critical for confirming stable transgene integration.

Additional Information:

• Probe 1 and Probe 3 - Targets the left flank and gene region, but combining them alone will not detect the right flank.

• Probe 2 - Targets the right flank, but it doesn’t help detect the left flank for identifying integration events across both regions.

• Probe 1 and Probe 4 - Targets left and right flanks but doesn’t consider the gene region.