Orientation of a DNA Insert

Ascertaining the Orientation of a DNA Fragment Insert

This is a preliminary to understanding the science behind the technology presented in kits such as those provided by Fotodyne Inc., and its technical assistance by Emily Devine.

Print out this overall figure and follow along!

Imagine that you have two solutions.
One vial contains a solution of identical plasmids that were digested with a restriction enzyme that clipped it at a specific site that was once a complete shaded box (). We could name the specific enzyme 'ase. After digestion, the plasmids are now identical open circles with -ends. The plasmid also has two other restriction sites on it - both the same, and those are denoted as ”. The other vial contains a solution of fragments of DNA derived from some creature such as a frog. Click here for how these fragments were derived. This fragment has ends that are the products of hydrolysis by a restriction enzyme we shall give a symbolic name ”'ase. Near one end there is another restriction site shown as a shaded box (). The arrowhead is to show you that the fragment is not a palendrome but is "polar", meaning that its DNA message is, like a book, has is a beginning and an ending.

Imagine that you have two solutions.

One vial contains a solution of identical plasmids that were digested with a restriction enzyme that clipped it at a specific site that was once a complete shaded box (). We could name the specific enzyme 'ase. After digestion, the plasmids are now identical open circles with -ends. The plasmid also has two other restriction sites on it - both the same, and those are denoted as ”.
The other vial contains a solution of fragments of DNA derived from some creature such as a frog. Click here for how these fragments were derived. This fragment has ends that are the products of hydrolysis by a restriction enzyme we shall give a symbolic name ”'ase. Near one end there is another restriction site shown as a shaded box (). The arrowhead is to show you that the fragment is not a palendrome but is "polar", meaning that its DNA message is, like a book, has is a beginning and an ending.

Were you to mix the vials' contents and add ligase to insert the zillions of fragments into the plasmids, those fragments would insert with equal probability in either direction. Hopefully you can see in the two large circles that they have done just that (the fragments inserted between the two -restriction sites and form the bottoms of the large circles).

The instructor will give you one or the other of these two large circles in a small vial. It is your job to ascertain the orientation of the fragment in the plasmid. This you do, by separately digesting both plasmids with "”'ase", and then using some technique that sorts the sizes of DNA fragments. The most commonly used sorting technology is electrophoresis.

The left circle would digest into three fragments: one short and two mediums
The right circle would yield the same short fragment, another short one and a very long fragment.

REMEMBER: only concern yourself with the lengths between the sites.

All those who see how the orientation can be ascertained, get up on your stools and chairs and whoop it up!

Okay, here's a quiz! Which one of the three electrophoresis gels fits best with the above theory.

The answer is on the lefthand bottom corner of this other page. There, click the answer to return to this place.

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The left circle would digest into three fragments: one short and two mediums	The right circle would yield the same short fragment, another short one and a very long fragment.
REMEMBER: only concern yourself with the lengths between the sites.