In this chapter we focused on how specific DNA sequences...

Question

In this chapter we focused on how specific DNA sequences can be copied, identified, characterized, and sequenced. At the same time, we found many opportunities to consider the methods and reasoning underlying these techniques. From the explanations given in the chapter, what answers would you propose to the following fundamental questions?

In a recombinant DNA cloning experiment, how can we determine whether DNA fragments of interest have been incorporated into plasmids and, once host cells are transformed, which cells contain recombinant DNA?

Accepted Answer

Hi, everybody. Let's take a look at this practice problem together which of the following techniques may be used to identify transform cells that have recombinant D N A. So for this question, I am using our D N A as shorthand for recombinant D N A. Now recall that recombinant D N A is engineered in a lab and it's done by combining two fragments to create a hybrid that wouldn't be found in normal genomes. So what technique identifies those transformed cells with recombinant D N A? Is it a blue white screening be antibiotic selection? See polymerase chain reaction or D all of the above. So the answer is actually all of the above. But let's go ahead and discuss each of these techniques a little bit further. We're going to start with blue white screening. Now in blue white screening, the recombinant D N A has the LAC Z JEAN and this is the gene for beta galactose sides growing the bacteria on special medium, results in colonies being either blue or white depending on whether they have the recombinant D N A. Now white colonies are the transformed cells and the blue colonies would be those that are non transformed. Now to discuss antibiotic selection, the recombinant D N A in this method is a plasmid with an antibiotic resistance gene. If cells are put into a medium with the corresponding antibiotic, the gene gives resistance to only transform cells will survive and grow in that medium since they have the resistance antibiotic resistance gene on the recombinant D N A. The last technique is PCR. Now recall that PCR uses primers and we can design the primers to target the recombinant D N A and that will amplify the DNA regions that contain the recombinant D N A. And so this is going to confirm the presence of recombinant D N A from the cells that samples were taken from. Therefore, it can identify the transformed cells and confirm them. So the answer is all of the above can be used to identify transform cells. Alright. Everybody I hope you found this helpful and I'll see you soon for the next practice problem.

Key Concepts

Recombinant DNA Technology

Selection Markers

Molecular Cloning Techniques

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