You have isolated another cDNA clone of the CRABS CLAW gene from ...

Question

You have isolated another cDNA clone of the CRABS CLAW gene from a cDNA library constructed using the vector shown in Problem 18. The cDNA was directionally cloned using the EcoRI and XhoI sites. You sequence the recombinant plasmid using primers complementary to the T7 and T3 promoter sites flanking the MCS (the positions of these sequences are shown in the figure in Problem 18). The first 30 to 60 bases of sequence are usually discarded since they tend to contain errors.

Can you identify which sequence portions are derived from the vector (specifically the MCS) and which are derived from the cDNA clone?

Accepted Answer

Hi, everyone. Welcome back. Let's look at our next question. It says a researcher wants to express a human insulin gene in bacteria using a plasmid vector with a multiple cloning site. M CS. The M CS contains several unique restriction enzyme recognition sites. The researcher plans to insert the insulin C DNA into the M CS of the desired location. What is the purpose of using the M CS in this context? Choice A to ensure that the bacteria can take up the plasma vector. Choice B to provide a location for the bacterial promoter to initiate transcription to c to allow for easy insertion of the insulin C DNA into the plasma vector or choice D to prevent the expression of other genes in the plasma vector. Well, this question looks a little bit long. Um but it's really quite straightforward when we think about inserting genes into a plasma vector for the sake of putting them in a bacteria and expressing the gene. The M CS is that location. As our question tells us of multiple restriction enzyme sites or re sites. And it has just one purpose which is to make a convenient spot where you can insert your desired gene, you cut the plasmid with the restriction enzyme, cut your desired gene with the same enzyme. They have matching sticky ends and can easily come together to make insertion simple. Um And the purpose of the course, the multiple restriction enzyme sites is you've got lots of possible enzymes with which you can cut your gene. So depending on what your gene sequence is, you have the possibility of using a bunch of different enzymes since you need to find obviously a matching restriction site in the gene of interest. So we're going to look at choice C here to allow for easy insertion of the insulin C DNA into the plasmid vector. So that was pretty simple, pretty straightforward. Uh If you like, we'll just go through the other answer choices to see why they're not correct choice. A says to ensure that the bacteria can take up the plasma vector. Well, this doesn't involve the restriction enzyme sites. That's just that process of transformation, either using a heat shock or electroporation. Um but doesn't have any involvement with the M CS Choice B to provide a location for the bacterial promoter to in initiate transcription. Uh No, that wouldn't be the M CS. The gene will have its own promoter, the plasma has promoters, uh the M CS is not a promoter location. And then finally choice D to prevent the expression of other genes in the plasmin vector. That isn't not the purpose of the M CS. It doesn't uh that it's just that site of insertion, the multiple sites mean it's very flexible, allows for cutting your desired gene with different enzymes. So choice d not our answer. The purpose of using the M CS when we want to insert our insulin gene into bacteria is choice C to allow for easy insertion of the insulin C DNA into the plasmid vector. See you in the next video.

Key Concepts

cDNA Cloning

Restriction Enzymes and MCS

Sequencing and Error Management

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