Restriction Enzymes Resource

2.6 Digestion of Restriction Sites Close to the End of Linear DNA

1. Multiple Digests
2. PCR Products
3. References

In order to recognize and cleave their recognition sequence, most restriction enzymes need some flanking DNA. Because of this it can be difficult to achieve complete digestion of PCR^(a) products that have restriction sites engineered near the end of a primer or to perform double digests using two enzymes that cut at sites close to each other in a polylinker region. Such digestions may be improved by using long (16-hour) incubation times.

A. Multiple Digests

When performing multiple digests within a polylinker region, it is important to determine if the sites overlap such that cleavage at one site will destroy another. For example, the sequence below contains both a Kpn I (GGTAC/C) and a Sma I (CCC/GGG) site.

...NNNNNGGTACCCGGGNNNNN...
...NNNNNCCATGGGCCCNNNNN...

If this DNA is first digested with Kpn I, it will leave the following sequence, which cannot be digested with Sma I.

...NNNNNGGTAC     CCGGGNNNNN...
...NNNNNC     CATGGGCCCNNNNN...

Alternatively, if the DNA is first digested with Sma I, it will leave the sequence shown below, which can be digested with Kpn I, although there may be problems due to a lack of flanking bases.

...NNNNNGGTACCC    GGGNNNNN...
...NNNNNCCATGGG    CCCNNNNN...

Studies by Kaufman and Evans (1), and Moreira and Noren (2) show the efficiency of digestion of polylinker regions with a variety of enzymes. This data can be used to help determine the order in which two enzymes should be used for the most efficient multiple digests, or to predict whether enzymes will work effectively in a double-digest. Care must be taken when applying the conclusions from these publications to the digestion of PCR products because the majority of the ends left by restriction enzymes have 2-4 base 3´ or 5´ overhangs. Generally, PCR products are either blunt ended (if a proofreading thermostable polymerase is used) or contain a single 3´ overhanging base (if a non-proofreading enzyme is used).

B. PCR Products

In general, the addition of 2-6 extra bases upstream of an engineered restriction site in a PCR primer will greatly increase the efficiency of digestion of the amplification product, but this is dependent on the enzyme used. Table 2.6 shows the results of a study where the ability of restriction enzymes to digest various PCR products was tested (3). PCR products in which the first base pair of the restriction site was flush with (0), or 1, 2, or 3 base pairs away from the end of the fragment were tested with a variety of enzymes.

Table 2.6. Ability of Restriction Enzymes to Cut PCR Products that have Engineered Restriction Sites Near the End of the Fragment.

Purified PCR fragments (10-50ng) were digested at least twice with 0.5 units of restriction enzyme in 10µl of the appropriate reaction buffer for 45 minutes. Digestion is indicated as follows: cleavable (+), not cleavable (–), and not reproducible (±). Table 2.6 reproduced by permission of Eaton Publishing.

The addition of upstream bases to PCR primers is not the only method used to improve digestion efficiency. A number of protocols have been proposed to improve digestion including proteinase K treatment to remove any thermostable polymerase that may be blocking the DNA, end-polishing with Klenow or T4 DNA Polymerase and the addition of spermidine. However, none of these methods have been shown to improve cloning efficiency significantly (4,5).

An additional drawback to the incorporation of restriction enzyme sites in PCR primers is that it can be quite difficult to resolve digested PCR products from those that remain uncut. This can be overcome by the addition of fluorescent tags at the 5´ ends of the primers prior to PCR. This allows identification of products that have been cut successfully because the label is lost upon digestion (6).

An alternative method that has been used successfully to improve digestion of PCR products is to concatamerize the fragments after amplification (1,5). This is achieved by first treating the cleaned up PCR products with T4 Polynucleotide Kinase (if the primers have not already been phosphorylated). The ends will already be blunt if a proofreading thermostable polymerase such as Pfu^(b) was used or may be treated with T4 DNA Polymerase to polish the ends if a non-proofreading polymerase such as Taq^(b) was used (5). PCR products are then ligated with T4 DNA ligase. This effectively moves the restriction enzyme sites away from the ends of the fragments and allows efficient digestion.

1. Kaufman, D.L., and Evans, G.A. (1990) Restriction endonuclease cleavage at the termini of PCR products. BioTechniques 9, 304.
2. Moreira, R.F., and Noren, C.J. (1995) Minimum duplex requirements for restriction enzyme cleavage near the termini of linear DNA fragments. BioTechniques 19, 56.
3. Zimmermann, K. et al. (1998) Digestion of terminal restriction endonuclease recognition sites on PCR products. BioTechniques 24, 582.
4. Jung, V. et al. (1990) Efficient cloning of PCR generated DNA containing terminal restriction endonuclease recognition sites. Nucl. Acids Res. 18, 6156.
5. Jung, V. et al. (1993) Cloning of polymerase chain reaction-generated DNA containing terminal restriction endonuclease recognition sites. Meth. Enzymol. 218, 357.
6. Yamaguchi, K. et al. (1994) Fluorescent primers allow direct confirmation of restriction enzyme cleavage of PCR products. BioTechniques 17, 649.