You have identified a 0.80-kb cDNA clone that contains the entire...

Question

You have identified a 0.80-kb cDNA clone that contains the entire coding sequence of the Arabidopsis gene CRABS CLAW. In the construction of the cDNA library, linkers with EcoRI sites were added to each end of the cDNA, and the cDNA was inserted into the EcoRI site of the MCS of the vector shown in the accompanying figure. You perform digests on the CRABS CLAW cDNA clone with restriction enzymes and obtain the following results. Can you determine the orientation of the cDNA clone with respect to the restriction enzyme sites in the vector? The restriction enzyme sites listed in the dark blue region are found only in the MCS of the vector.

Accepted Answer

Welcome back. Everyone. Here's our next problem. It says the same restriction enzymes are often used to cut both the plasma DNA and the gene insert, which are then joined together with the help of an enzyme known as choice A kilo case B reverse transcriptase, C DNA polymerase or D DNA ligase. So when we're inserting a gene into a plasmid, we cut them both with the same restriction enzyme. When you do this, you generate what are called sticky ends. So instead of having your DNA strand cut ending evenly in the same place on both strands, it means that the strands end at different bases leaving overhanging ends. And then of course cutting both the plasmid and the gene you want to insert with the same restriction enzyme means that these overhanging ends match each other and just need to be joined together. And when we talk about joining DNA together, joining together breaks and DNA, we're looking at choice D DNA ligase as the enzyme we want. Let's just take a look at other answer. Choices. Choice A helicase. We know from that name, that presence of that word for helix heli cases. Unwind R, DNA double helix as part of the DNA replication process so that you can separate the two strands but not what we're looking for. Choice B. Reverse transcriptase generates a DNA strand from an R N A sequence. That's why it's reverse transcript A, since it's reversing the process of transcription, but not what we're looking for. And finally DNA polymerase is what synthesizes a DNA strand in the process of DNA replication, but also not what we're looking for. So when we have cut two pieces of DNA with the same restriction enzyme and we want to join them together, that enzyme that does, that is choice D DNA ligase. See you in the next video.

Key Concepts

cDNA Cloning

Restriction Enzymes

Vector and Multiple Cloning Site (MCS)

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