Briefly describe two different methods for inserting foreign...

Key Concepts

Restriction Enzyme Cloning

This method involves cutting both the plasmid and the foreign DNA using specific restriction enzymes to create complementary sticky or blunt ends, followed by the use of DNA ligase to join the DNA fragments. Its strengths include high specificity and the relative ease of use with many enzymes available. However, it is limited by the need for appropriate restriction sites in both the plasmid and the insert, and may introduce unwanted sequences or require additional steps if the sites are not conveniently located.

Recombination-Based Cloning

Recombination-based methods, such as Gibson Assembly or in vivo homologous recombination, allow the insertion of foreign DNA into plasmids without the reliance on specific restriction sites. These techniques use overlapping regions between the insert and vector to enable precise and seamless assembly. The advantages include flexibility in design and the ability to assemble multiple fragments simultaneously, while the drawbacks can include the need for careful primer design, potentially higher costs for reagents, and sensitivity to reaction conditions which might affect efficiency.

Transcript

00:01 There's many different methods to getting dna or foreign dna into plasmids.

00:07 And these were developed basically to just help insert dna in different situations because there was not one catch -all method of doing it.

00:19 Now, the most generic way, and i say generic, because if you look in any textbook in your most elementary, it's the only one they're going to talk about.

00:28 And then you're more advanced is the first one they talk about.

00:30 With a plasmid, in order to get new dna into a plasmid, you know the dna sequence of your plasma.

00:38 That's a known known.

00:41 And knowing that, you can use restriction enzymes to help cut out pastes in your plasmen.

00:48 So you can have a restriction enzyme that cuts out that area right there, and i'm going to mark it in blue.

00:55 And then inside a solution, you can also have a sequence that has the same recognition site.

01:07 We shouldn't make that green because then they may get confused.

01:10 Let's make it orange.

01:12 And so what happens is when you have a restriction enzyme digest the plasmine is going to cut out this reason.

01:22 Doesn't technically make it disappear, but it's going to remove it from these spots here, these sticky ends here.

01:29 And what's going to happen is when the dna repairs itself, it can now pick up and this doesn't happen every time, but it happens with enough efficiency to be a useful practice.

01:39 It cannot pick up that new dna sequence and then that new sequence right here...

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