Restriction Enzyme Digestion

[hsip_15]

Hi to all,

I am currently attempting to do digestion of my plasmids now.

However, I am wondering on how the enzymes really work since my basic in Biology is weak.

The enzymes have a specific site to cleave the genes in the plasmids at the multicloning site. The enzymes are resembled in the products' website with a 5' to 3' and 3' to 5'. Therefore, it seems like they are a string of DNA. Since DNA in plasmids are in double helical forms, how does the enzyme actually cut the plasmids and what will become of the plasmids after that? open circular and nicked are still in double helical form?

Thank you.

[kmunson779]

Think of double-helix DNA as two sticky strings (say, one blue and one red) twisted together. To make a plasmid, take the twisted string and tie the ends of the red strings together, then tie the ends of the blue string together. You'll have a circle made up of two circular pieces of string that are twisted and stuck together.

Supercoiled plasmid occurs when the red and blue are twisted together so tightly that they start to kink up (Seriously, take a piece of string and twist it, you'll see what I mean.) If a plasmid is nicked, imagine untying the knot in the red string. Now it can untwist around from the blue string and release some of the kinks. It's still double-helical, since the two strings are stuck together (modeling the base pair bonding), but it's in an "open" or "relaxed" conformation.

Here's where my model breaks down: each of the strings has a direction, and the two strings' directions are opposite. That's the 5'/3' designation.

Enzymes usually recognize a palendromic DNA sequence (for example, EcoRI cuts 5'-G'AATTC-3'. Because it's palendromic, when you have the double stranded DNA, you have the same sequence reading either way:

5'-G'AATT C-3'
3'-C TTAA'G-5'

(if you take your monitor and flip it upside down, you'll see that this sequence looks the same either way you look at it)

So, the enzyme recognizes the double-stranded sequence, and cuts each strand where I've put the tick mark, giving you a linear plasmid with the ends:

5'-G         AATTC-3'
3'-CTTAA         G-5'

The string is still twisted and stuck together, but it's back to being a string rather than a circle.

Does this help at all? Or make any sense to you? Or was this not your question?

[moleculardude]

Great post Kmunson. I learned a few things from that too!

Yes open circular and nicked are both double helical. The enzyme only affects the ends of the DNA molecule, not the structure.

Although, I am not sure what happens to the single stranded DNA ends. I guess they are not double-helical as they are single stranded.

See Kmunsons post above. The unpaired nucleotides on the ends as you see will not be double helical as they are unpaired ( hence no double helix).

Actually the only one restriction cut that has double helix ends is the BLUNT cut where it is like this:

GACTACC
CTGATGG

after cutting

GAC TACC
CTG ATGG

[kmunson779]

The unpaired nucleotides on the ends as you see will not be double helical as they are unpaired ( hence no double helix).

Right. Sorry I missed that -- the EcoRI ends are single-stranded DNA, called "sticky ends" since they want to base pair with a complementary single strand...

[admin]

Hey Kmunson,

I never thought of that either until moleculardude pointed it out.

thanks moleculardude

[hsip_15]

kmunson779 and molecular dude ..

Thanks a lot. that helped clear some haze in my mind.