b) BclI cleaves after the first T:
3’ TGATCA 5’
5’ ACTAGT 3’
Does cleavage by BclI result in a 5’ or 3’ overhang? What is the sequence of this overhang? [1]
To determine whether cleavage by BclI results in a 5' or 3' overhang and the sequence of the overhang, we need to analyze the recognition sequence of BclI.
The recognition sequence of BclI is 5'...TGATCA...3', which means the enzyme cleaves after the first T nucleotide in this sequence.
Given the recognition sequence 3' TGATCA 5', we can identify the specific site of cleavage. BclI cleaves between the first T (T) and the following G (G), resulting in the following fragments:
- 5' TGAT (cleavage site) CA 3'
- 3' ACTA (cleavage site) GT 5'
Now, let's determine the overhang by looking at the two fragments:
- The first fragment is TGATCA, and since the cleavage occurs after the T, the overhang is on the 5' end. Therefore, the overhang is a 5' overhang.
- The second fragment is ACTAGT, and since the cleavage occurs before the G, there is no overhang on the 3' end.
To summarize:
- Cleavage by BclI results in a 5' overhang.
- The sequence of the overhang is TGAT.