Wednesday, April 7, 2010

My Research Explained: DNA Sticky Ends

Last time I talked about DNA structure and sequence. I told you that the sequence is important in lots of ways and one of those reasons comes in the form of protein recognition sites, sequence of DNA that proteins can bind to and perform actions. One of the key proteins in my experiments is known as a Restriction Enzyme and has the ability to cut double stranded DNA at a specific site, its recognition site.

There are lots of Restriction Enzymes and each one cuts (or digests) the DNA differently. Some recognize 6 basepairs (bp) and cut internally to that site, some recognize 6 specific bases but also a several in the middle with no discretion, some cut after the recognition site, some cut and leave an overhang of DNA, while others cut and leave a blunt end. I'm interested in the ones that leave an overhang.

Let me use a specific enzyme to describe the process. XhoI (magically named so in my head) is a restriction enzyme that recognizes the sequence CTCGAG. This sequence is special because it's compliment is the exact same thing making it palindromic. The enzyme will cut the DNA just after the first C in both the top and bottom strands. So after digestion you will have two strands that look like this:

...C TCGAG...
...GAGCT C...

Notice that one end of one strand is longer than the end of the other strand. That is what I am referring to when I say overhang. This overhang can be attached to a complementary strand of DNA, giving this piece of DNA a sticky end! Because it is palindromic the end could fold up and stick to itself (the T at the very end could stick to the first free A). In my research I use enzymes that cut so that the sticky end isn't palindromic and has an overhang that isn't nearly as common. More on this next time.

In a process known as ligation, an enzyme known as DNA Ligase has the ability to attach two complementary strands of DNA together making one perfect strand with no blemishes (well most of the time) or anything. So if my overhang isn't palindromic and I don't have a common sequence overhang I can put a bunch of different DNA into reaction and end up with a fairly specific product and that is just taking no precautions. If I plan the process well I could get nearly 100% of exactly the DNA sequence that I want. Now, what sequence would I want anyways?

1 comment:

Steve Koch said...

Great explanation!

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