Restriction Enzymes

[laczlacylaci]

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I am bit confused about restriction enzymes and how they recognize to perform their function.

Situation 1) A bacteria cell is infected by a bacteriophage, which injected some viral progeny into it. Restriction enzymes recognize the viral DNA due to its unmethylated palindromic sequence. Once recognized, the viral DNA is somehow degraded. The restriction enzymes don't "attack" the bacterial DNA since it is methylated. [concept learned: the mechanism for bacterias to eliminate foreign genetic components: restriction enzymes degrade them by recognizing unmethylated palindromic sequences.] (or recognizing unmethylated/methylated palindromic sequences?)

Situation 2) Let's say we wanted to clone gene A (gene of interest). I read that we could potentially extract the bacterial DNA out and use a restriction enzyme (ie. EcoR1) to cleave at a palindromic sequence in order to expose the sticky ends. (but isn't this bacterial DNA methylated? Therefore, how did the EcoR1 cleave it?)
As for gene A, we can do the same and expose the sticky ends. We can then combine these two, put it back into the host cell/original bacteria and replicate.

My confusion is how does restriction enzymes exactly recognize. What am I missing? I am assuming different restriction enzymes can cleave (unmethylated &/or methylated DNA), depending on the restriction enzyme?
Also, any questions that refer to this topic is appreciated for practice. Thank you!

 

[DoctorCakes]

Restriction enzymes are pretty complex proteins therefore they do not always get blocked out by methylation. This function varies throughout different strains, an EcoR1 in E.coli can recognize and cut methylated sites while another strain of E.coli EcoR1 may not. All enzymes are different and I would not look too much into it. It seems you have a good understanding of restriction enzymes that should suffice

 

[wizzed101]

1) Correct.
2) You have to understand that genetics is not an exact science in the sense that you cannot pick exactly one DNA sequence and insert it into exactly one plasmid and insert it into exactly one bacterium. The general strategy is to throw everything at the wall and see what sticks. After you learned this (like I did), every procedure makes sense!

What really happens is the gene of interest will be first isolated by its mRNA (into cDNA) and then a special enzyme is used to add sticky ends. The resulted sequence will be inserted to engineered plasmids. Then a lot of those plasmids will be used to transform colonies. Restricting conditions will be used to select bacteria that had uptaken the plasmid. In fact very very few of the original bacteria will because bacteria in general don't like plasmids as you can imagine! They have lots of mechanisms to degrade them. But that's ok. Like I said, the strategy is to throw plasmids at them and select the few that accept it.

Plasmids can stay as plasmid or be integrated into host genome (rare- but like I said, it doesn't matter if it's rare!) by crossing over.

 

[laczlacylaci]

Restriction enzymes are pretty complex proteins therefore they do not always get blocked out by methylation. This function varies throughout different strains, an EcoR1 in E.coli can recognize and cut methylated sites while another strain of E.coli EcoR1 may not. All enzymes are different and I would not look too much into it. It seems you have a good understanding of restriction enzymes that should suffice

1) Correct.
2) You have to understand that genetics is not an exact science in the sense that you cannot pick exactly one DNA sequence and insert it into exactly one plasmid and insert it into exactly one bacterium. The general strategy is to throw everything at the wall and see what sticks. After you learned this (like I did), every procedure makes sense!

What really happens is the gene of interest will be first isolated by its mRNA (into cDNA) and then a special enzyme is used to add sticky ends. The resulted sequence will be inserted to engineered plasmids. Then a lot of those plasmids will be used to transform colonies. Restricting conditions will be used to select bacteria that had uptaken the plasmid. In fact very very few of the original bacteria will because bacteria in general don't like plasmids as you can imagine! They have lots of mechanisms to degrade them. But that's ok. Like I said, the strategy is to throw plasmids at them and select the few that accept it.

Plasmids can stay as plasmid of integrated into host genome (rare- but like I said, it doesn't matter if it's rare!) by crossing over.

This makes sense! Thank you for the clarification!