Restriction Enzyme Digestion of Recombinant Plasmid DNA

Overview and background:

Following the cloning of a new gene or fragment of DNA, there are a number of ways to confirm that you have successfully completed the cloning reactions, before proceeding with downstream protocols. It is highly desirable to be certain of the integrity of your recombinant clone prior to performing lengthy and costly experimental procedures such as transfection and the analyses that would follow. Although your gene was already subcloned into its plasmid vector prior to your receiving it, we will still proceed with confirmation of its integrity.

We will use restriction digestion with specific enzymes (endonucleases), in addition to a separate ‘validation’ step by polymerase chain reaction (PCR), to confirm that the expected inserts (genes) are actually present in your plasmid vectors. Results of these tests will be analyzed by agarose gel electrophoresis, at which time we will compare your results with the ‘expected’ results that were derived from the known sequences (and sizes) of your genes.

Using web tools described below, each team will choose one or two different restriction enzymes with which to digest a small aliquot of their recombinant plasmids. The choices should be turned in (by email) to SAC by ~5:00 PM on the following Friday (10/10), allowing time for ordering of enzymes if necessary. The following lab session (10/14), restriction digest reactions with your chosen enzymes will be carried out, followed by agarose gel electrophoresis.

Restriction enzymes are proteins produced by bacteria that cleave DNA at specific, short (usually ~6 base-pair) sequences, making a cut in each phosphate-sugar backbone of the double helix without damaging the bases. For example, the commonly used enzyme Eco R1 (named after its E coli host) makes a “staggered” cut in the following (palindromic) sequence, wherever it occurs:

G A A T T C                          5’…G                                5’…AATTC…3’

C T T A A G                          3’…CTTAA…5’        +                      G…5’

A “palindrome”, in this context, refers to a nucleotide sequence with a symmetry that causes it to be read the same way in the forward or reverse (complementary) direction, i.e. “GAATTC” and

The cut DNA then separates into fragments, sometimes with “sticky ends” or “overhangs” as seen above (“5’ overhang” in this case). It should be obvious that a second DNA fragment with the same “sticky ends” (generated by the same enzyme) could hybridize to (bind to) the ends illustrated above, and all of the DNA fragments could then be ligated together (enzymatically) to create a recombinant DNA fragment.

In their native bacterial host cells, restriction enzymes provide protection against foreign invading nucleic acid sequences (similar recognition sequences in the host genome are protected from digestion by methylation). For scientists, these enzymes are very useful for cloning procedures as illustrated above, and also for detecting/verifying the presence of specific sequences within a piece of DNA. Digestion of DNA by currently available enzymes is a fast, relatively simple procedure and as such, these enzymes are extremely useful tools.

When a DNA sequence is known, the locations of recognition sequences for various restriction enzymes can be predicted, sometimes with the assistance of programs such as “NEB Cutter”, available through the New England Biolabs website.

The resulting sizes of DNA fragments generated, visible on agarose gels, will be compared to the expected fragment sizes, thereby verifying that you have (or do not have) the specific insert you were thought to have.

Choosing your restriction enzymes:

The goal is to use restriction enzyme digestion to distinguish your recombinant plasmid (Gene A, B6, or F) from the ‘empty vector’ plasmid pCMV6_AC_tGFP. Try to choose enzymes that will yield two or three well- separated fragments from your clone2, and make sure that this pattern of fragments would clearly distinguish your clone from ‘empty vector’ digested in the same manner.

To design this restriction digest, go to NEBcutter:

http://tools.neb.com/NEBcutter2/

In Bb, open the word document that contains the DNA sequence for your gene. A=Rassf1A

B6=ITGB6, or beta 6 integrin

F=Fas

Select/copy the entire sequence of your gene/plasmid. (Do not include the color key at the bottom).

Paste the sequence into the empty box on NEB cutter. Click “circular” (optional: name the sequence where prompted, to allow you easy access to this worksheet) Submit

You now have a diagram showing the restriction enzymes that cut this plasmid/gene construct once (“single cutters”). The grey bar designated ‘a” represents your hybrid gene/tGFP (or gene/mGFP). For each gene, the enzyme AsiSI marks the 5’ boundary of the gene sequence; the enzyme Mlu I marks the 3’ boundary of the sequence for the gene itself (the insert), however remember that the GFP ‘tag’ gene is downstream of this site. Note these boundaries, because you will want to choose enzymes between them (and excluding those ‘boundary’ enzymes).

There is more than one correct strategy for this procedure, but I have found the simplest way to be the following (and it allows for a single enzyme to accomplish the task):

After visualizing the ‘single cutters’ and noting the boundaries, proceed to the double (or triple) cutter diagram by selecting “2 cutters” OR “3 cutters” within Display. Now locate an enzyme that cuts somewhere within the boundaries of the ORF itself (and not GFP, if possible); note the location of its second and third, if applicable) recognition site(s) by holding the cursor over it, and locating the red underlined areas. Provided this other site(s) is somewhere in the vector backbone (outside of “a”), then proceed to visualize the expected fragments as follows.

Go to Custom Digest. Select your enzyme from the list provided and hit the green Digest box. You can hit the “View Gel” tab to see the sizes of the products (fragments) and how they would appear in an agarose gel. If they are distinguishable in the gel (as opposed to being too close together), then your enzyme is most likely a feasible choice.

In this same manner, visualize the fragments you would obtain if the vector (pCMV6_AC_tGFP) was cut with this enzyme. Most likely, you will obtain one less fragment and/or fragments of differing sizes, compared to your recombinant clone.

Important: Try to obtain fragments that are at least 500 bp apart in size, and larger than ~200 bp.If you end up choosing a combination of two different enzymes to accomplish this task in a ‘double digest’ (as opposed to a single enzyme), you must find out if the enzymes are compatible, that is, if they can be used in a digest reaction together, under similar conditions. Not all of them can, due to different buffer requirements. To determine this:

Go to this page:

http://www.neb.com/nebecomm/DoubleDigestCalculator.asp?

Insert the enzyme names and note the conditions (buffer and temperatures and additional cofactors, such as BSA) needed for a double digest. If a double digest (i.e., both enzymes together in one tube) is not feasible, please choose another pair (may keep one out of the original two). Time will not allow for us to do sequential digestions.

Bottom line: Whether you choose one or two different enzymes for this digest reaction: ideally, one of the enzymes should cut the plasmid DNA within the ‘gene of interest’ coding region, which will show up as the ‘upper half’ of the major ORF (“a”) in the NEB cutter diagram. (Lower half of that major ORF will be GFP gene.) Other ‘cuts’, whether by the same or a different enzyme, should be in the vector ‘backbone’.