Why are restriction enzymes palindromic?

Oooo my best guess during lecture seems to be supported on the interwebs! Not all REs have palindromic recognition sites, but most of the ones that do operate as homodimers (this means that two molecules of an identical protein have to associate with one another for it to function). Each half of the dimer recognizes the same sequence, leading to a double-stranded break… remember, the evolutionary function of these enzymes is to chop up invading phage DNA, and this requires double stranded breaks. The homodimeric RE is more efficient than a heterdimeric RE because it only requires ONE gene to be present in the genome and expressed.

When is it that we don’t need alkaline phosphatase to treat the digested plasmid?

I am going to assume that the plasmid is double-digested by two REs and creates incompatible, sticky ends, while the DNA fragment is amplified by PCR and pre-digested before ligation.

Treat your vector with AP if it can re-anneal due to the presence of compatible ends AND your insert has 5’ PO4s on both ends. DO NOT treat with AP if your vector cannot re-anneal OR your insert does NOT have 5’ PO4s on both ends. It’s ok, but not required to treat your vector with AP if the ends are not compatible… when you run a double digest, there is always a chance that one of the enzymes will not completely cut every single molecule in the tube, leaving compatible ends even when you intend for them to be incompatible.

How do you identify restriction enzyme sites on DNA?

There’s an app for that… and for most things having to do with restriction enzymes. Here’s a link to all of NEB’s apps. You’d want “NEBcuttter” to find a list of restrictions enzymes that cut a specific sequence. https://www.neb.com/tools-and-resources. If you are using Snapgene or CLC Workbench as cloning tools, these can be set to show restriction enzyme sites.

Do we have to learn the restriction enzyme sequences?

No! We will not ask you to memorize any DNA sequences or RE sites.

What is a [restriction enzyme] buffer and what does it do?

Restriction enzyme buffers are salt solutions that support optimal enzyme activity. For example, ThermoFisher’s 10X Buffer B for restriction enzymes contains 100 mM Tris-HCl, 100 mM MgCl2, and 1 mg/mL BSA and is pH 7.5 at 37C.

Is there a clearer method to solving these enzyme problems? A step-by-step guide? I really don’t understand the information on the worksheet. I need a better understanding of the problems.

In addition to the first page on the in-class worksheet handout, and the extra practice folder on Moodle, I’ll direct you to NEB’s protocol for optimizing restriction endonuclease reactions (link below). We’ll go over another problem as review in Lecture 3 and you’ll get practice during this week’s lab. If it is still problematic, please arrange a time with me for office hours. https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions

I want to practice more on how to do calculations of reaction units. I’m still not clear on how to calculate concentration.

Since different restriction enzymes cut at different sites, we use empirically-derived “reaction units” to define activity. By definition, 1 unit (U) of restriction enzyme will completely digest 1 ug of substrate DNA in a 50 uL reaction in 60 minutes. However, it is standard practice to use 5-10U per 1ug DNA when actually running a reaction.

So, do we identify DNA sites based on features and restriction sites?

A restriction enzyme recognition site is the specific sequence of DNA that is recognized by a specific restriction enzyme (there are thousands of them). They’re essentially very small features (a feature is a sequence of DNA that has a function). If you want to locate all the restriction sites on a sequence of DNA, enter the sequence into NEBcutter (online) – it will report all the restriction enzymes in their database that can bind to your DNA and the specific locations where they bind.

Why will increasing the weaker enzyme be a solution to problem 3 on the handout?

By definition, 1 unit of restriction enzyme will completely digest 1 ug of substrate DNA in a 50 uL reaction in 60 minutes. A value of less than 100% for the “% Activity in NEB Buffer” means that that the enzyme will not be able to completely digest 1 ug of DNA. If you double the amount of enzyme available in the reaction, you double the number of enzyme molecules available to interact with DNA. This increases the likelihood of achieving complete digestion within 1 hour.

For more information on optimizing restriction endonuclease reactions, check out NEB’s protocol on the topic: https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions

Do restriction enzymes contain non-compatible ends?

The enzymes themselves do not have ends. Heading back to Lecture 3 where I re-addressed this point, “blunt”, “sticky”, “compatible” and “incompatible” are terms that describe one end of DNA or multiple ends relative to each other. A single end of dsDNA can be blunt or cohesive. This describes the bases on the end of the sequence; if blunt then there are no free base pairs; if cohesive then there are free, unpaired bases. Remember, base pairing involves hydrogen bonds between A-T and G-C groups. Regardless of the base composition, the 5’ end of DNA has a free phosphate group (PO4) and the 3’ end has a free hydroxyl (OH) group. Two ends of dsDNA can be compatible or incompatible – if compatible, it is either cohesive or blunt. Cohesive ends have bases that match up; blunt ends have no free bases, but still have a 5’ phosphate and 3’ hydroxyl group. Come to see me if this doesn’t clarify the point for you – it’s probably better drawn than written.

Restriction mapping. Could you go over using proper REs in restriction mapping for determining gene presence vs orientation?

The principle behind restriction mapping is that REs cut DNA at specific sequences. If you know the predicted sequence of a piece of DNA, you can identify what REs cut the sequence and where they cut. Gel electrophoresis can distinguish between DNA fragments of different sizes. So, restriction mapping works by designing a RE digestion strategy that will produce one unique fragment size pattern if the DNA has the expected sequence (presence and orientation) and other patterns if the sequence is not as expected.