Consider a jigsaw puzzle. While most of the pieces have a different picture on their surface, all pieces fit together in an interlocking pattern.
As unlikely as it may seem, restriction enzymes from different organisms can produce interlocking pieces of DNA – so called compatible cohesive ends (CCE). These are pieces of DNA, which fit together and can be ligated, creating a hybrid molecule.
It’s all about isoschizomers
The recognition sequences of type II restriction enzymes used in molecular biology are palindromic, e.g. symmetrical. Further constraints on the restriction enzymes are the requirements to recognize certain bases in certain positions of a palindrome. The result is a finite number of the solutions for DNA recognition and cleavage puzzle – a lot of different species have unrelated enzymes, which recognize the same sequence (they are called isoschizomers).
Of course, by definition, isoschizomers often produce compatible cohesive ends. However, unrelated enzymes with different recognition sequences can also produce compatible ends, which can be ligated together. Have a look at Figure 1 in which two different DNA molecules, cleaved by Cla I and BstB I, respectively, can be ligated together. In this case the resulting, hybrid molecule can be further re-cleaved by the third enzyme, Taq I. Conversely, ends cleaved by Taq I are compatible with Cla I and BstB I and several other enzymes.
Figure 1. Example showing the generation of compatible cohesive ends.
Putting isoschizomers to work for you
You can see from Figure 1 that using CCE destroys both original recognition sequences.
So what’s the point in using CCE for cloning?
Well, sometimes it’s the only way to ligate, saving the necessity of a PCR.
More importantly, destruction of the restriction sites can be a useful diagnostic tool to distinguish between your vector self-ligation and the resulting construct containing the fragment of interest.
Identifying CCE
How would you know if your enzymes of choice produces CCE?
The simplest, low tech way is looking in the New England Biolabs catalog, which contains a lot of useful information in the last section. In the 2013-2014 edition the list of enzymes producing CCE, what they can be ligated to and how to re-cleave hybrid molecules is located on pages 312 – 314. Alternatively, NEB also provide this information on their website.
Let’s have fun with DNA jigsaw puzzle!
More 'DNA / RNA Manipulation and Analysis' articles
Generating a DNA vector library of single and/or compound mutants for a target protein can be a daunting task. If you’re lucky and work in a well-funded lab, you might outsource this process via gene synthesis. Most of us though, need to do it the old-fashioned way. Traditional QuikChange™ Traditionally, there are many steps you…
I always keep an ear open for helpful tips in the lab – those little tricks that can make your experiments faster, easier and better. Here are a few tricks I’ve picked up for trimming down the time it takes to do your cloning: Restriction digests Many digests are complete within 10 minutes of digestion…
Alternative splicing events often occur in a spatiotemporal manner, and some are regulated by alternative splicing regulators, with striking variation across tissue types and developmental stages. Alternative splicing events are often differentially regulated across tissues and during development, as well as among individuals and populations, suggesting that individual isoforms may serve specific spatial or temporal…
In Part I of AAV Production, I described how to produce crude (non-purified) AAV. In this article, I am going to tell you how to purify that crude prep. Virus purification is usually done by gradient ultracentrifugation. Two common methods involve gradients made from increasing concentrations of cesium chloride or iodixanol. A cesium chloride prep…
10 Things Every Molecular Biologist Should Know
The eBook with top tips from our Researcher community.
