Enzymatic Digestion

introduction

One of the critical steps in genetic engineering is enzymatic digestion, which employs restriction enzymes. Restriction enzymes cleave specific sequences of double-stranded DNA. The following technique can be used to facilitate the transfer of any DNA fragment, applicable when the restriction enzyme cleavage sites flank the desired gene, allowing for the creation of complementary sequences at suitable locations within a vector to enable the gene's incorporation into a plasmid.

Restriction enzymes (restriction endonucleases) are proteins that cleave DNA at (or near) specific recognition sites (refer to manufacturer catalogs or restriction enzyme databases). There are two types of restriction enzymes that act differently in how they cut the target DNA:

Blunt-end cleavage: These restriction enzymes cut both strands of the target DNA at the same point, resulting in blunt ends.

Sticky-end cleavage: These restriction enzymes cut both strands of the target DNA at different locations, generating 3′- or 5′-overhanging ends, typically 1 to 4 nucleotides long (referred to as sticky ends).

To clone or express a specific DNA sequence in a vector, both must be digested with two restriction enzymes that generate complementary ends. At least one of the restriction enzymes used should produce a sticky end to ensure that the specific gene is oriented correctly.

Subcloning via restriction enzyme digestion is a common laboratory technique. For the purposes of this tutorial, we will discuss how to transfer cDNA from one plasmid to another. However, this method can also be utilized for moving promoters, markers, or any other DNA sequences between plasmids.

Let’s assume you are starting a new project involving your gene of interest (YGOI). You may need to clone YGOI into mammalian cells. The challenge is that the only complete cDNA version (synthesized from mRNA sequence) available for YGOI is in a bacterial expression vector. By using subcloning, you can easily transfer YGOI to a mammalian expression vector.

The desired gene is flanked by two restriction sites, and the plasmid is separately digested with the same restriction enzymes. This results in complementary sticky ends on the plasmid and the target gene, allowing them to be ligated together.

Design (Selection of Restriction Enzymes)

Numerous DNA analysis tools, including the Addgene site, allow you to identify restriction enzyme cleavage sites within a specific sequence. When selecting restriction enzymes, choose those that:

Cleave both ends of your target gene without leaving a cut site in the middle.

Have their cleavage site located at the desired position in the plasmid, preferably at the multiple cloning site (MCS), without cutting elsewhere in the plasmid.

Ensure the placement of your gene in the recipient plasmid (be cautious not to express the antisense version of your gene!).

Position the cleavage site appropriately concerning the genes of interest for selecting plasmid-containing colonies and recombinant plasmid colonies (we will publish an article on colony selection in the future).

Ideally, you will find two different restriction enzymes for your subcloning. A single enzyme can also be used, but in that case, you would need to treat the recipient plasmid with a phosphatase and perform a specific enzyme digestion test to confirm successful cloning.

If you cannot find suitable restriction enzymes that meet these criteria, do not worry. You have other options, such as:

• Adding desired restriction enzyme sequences to both ends of the gene: You can use PCR-based cloning to append the restriction site sequences to the ends of your oligos. This allows you to generate a version of your target gene alongside restriction sites compatible with the MCS of the recipient plasmid. However, you still need to avoid restriction enzymes that cut within your target gene sequence.

• Adding desired restriction enzyme sequences to the recipient plasmid: Using annealed-oligo cloning, you can modify the MCS of your recipient plasmid.

  

The target gene is excised from the donor plasmid with two different enzymes. The recipient plasmid is digested with the same two enzymes, generating complementary sequences to ensure the target gene is oriented correctly within the recipient plasmid.

If you are fortunate enough to have various options for restriction enzymes that are suitable for your target gene and ensure correct orientation in the recipient plasmid, it is beneficial to determine if a set of enzymes can work in the same restriction enzyme buffer (for more information on restriction enzyme buffers, refer to New England Biolabs). Choosing enzymes that can function in a single buffer will save time in later steps.

Select restriction enzymes for both your donor and recipient plasmids. Since some DNA is lost during the gel purification step (post-plasmid isolation), it is important to digest a substantial amount of plasmid. We recommend using 1.5-2 μg of donor plasmid and 1 μg of recipient plasmid. It is also crucial to ensure the recipient plasmid is digested with both enzymes as thoroughly as possible.

If you intend to use only one restriction enzyme or a set of restriction enzymes that will generate complementary sticky ends or no free ends post-digestion, you must use phosphatase to prevent the re-ligation of the recipient plasmid. Depending on the phosphatase you choose, you should treat your digested recipient plasmid with phosphatase either before the ligation step or before the gel purification step. Typically, CIP (calf intestinal phosphatase) or SAP (shrimp alkaline phosphatase) is used.