Ligase catalyzes a reaction between the 5′ phosphate and the 3′ hydroxyl groups at the ends of DNA molecules. The enzyme calf intestinal phosphatase catalyzes the removal of the 5′5′ phosphate from DNA molecules. What would be the consequence of treating a cloning vector, before ligation, with calf intestinal phosphatase?

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Hi, everyone. Let's look at our next problem. What is the function of calf intestinal phosphatase in molecular cloning? Choice? A it enhances the activity of the ligase enzyme B, it promotes the ligation of DNA fragments into the vector. Choice C it D phosphors, the five prime end of DNA fragments leaving a hydroxyl group or choice D it Deos fours, the three prime end of DNA fragments allowing for efficient ligation. Well, calf intestinal phosphatase or C IP is used to prevent cloning vectors from relegating without the desired DNA insert. So you're trying to put your desired gene into the vector to be delivered. You don't want your vector to close back up after you cut it with the restriction enzyme without having a DNA inserted. Um because that reduces the efficiency of the process and causes a high level of background colonies which makes the whole process more difficult to eff effectively get your modified bacteria. So how does the C IP do that? Well, it, the answer is choice C it defoor relates the five prime ends of the DNA fragments of the vector. Gonna add that at the end of choice C leaving A hydroxyl group. So we kind of have a clue in the name of the enzyme. It's called a phosphatase. So, you know, it's doing something with a phosphate group so that can help point you in the right direction. So we know that choice C is our answer. We can eliminate choice D because it has the correct activity, this D phosphorylation. But the wrong end since choice D says it desolates the three prime end of the DNA fragments. So choice D is incorrect to A says it enhances the activity of the liga enzyme. Um That's not a correct description of what it does. It prevents relig of the vector. And finally, choice B says it promotes the ligation of DNA fragments into the vector. Well, it does in a sense, but that's not a good description since it prevents the vector from relegating without the DNA fragment being inserted, but it doesn't do anything to improve the ligation of the DNA fragment. So choice B is not the correct answer. So again, the function of the C IP enzyme in molecular cloning is choice C it D phosphors five prime ends of DNA fragments leaving a hydroxyl group. See you in the next video.

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Key Concepts

DNA Ligase Function

Role of 5′ Phosphate in Ligation

Effect of Calf Intestinal Phosphatase (CIP) Treatment