The most common issues when performing digestion reactions are caused by unexpected cleavage patterns. In this article, we have highlighted the most recurrent problems and probable causes to help you resolve your hurdles.
Below is the guide of what issues this article will be covering.
| Common issue | Probable causes |
|---|---|
| Incomplete or no digestion |
|
| Unexpected cleavage pattern |
|
Incomplete or no digestion
Inactive restriction enzyme
A restriction enzyme may lose activity due toimproper storage or handling.Here are solutions to help you prevent and address this issue.
Confirm theexpiration date, verify that the restriction enzyme has beenstored at -20°C, and check the temperature ofyour freezer(do not allow temperatures to exceed -20°C, asmultiple freeze-thaw cycles(more than 3 cycles) may result in reduced enzyme activity).
Test the enzymefor activity by setting up acontrol reactionwith 1 µg ofstandard control DNA(e.g., lambda DNA), where you know that the DNA quality is high and the expected banding pattern (Figure 1).
Figure 1. λ DNA digested with BamHI, 0.7% agarose, 5 cleavage sites
Avoid storing enzymes infrost-free freezersthat undergotemperature fluctuations. It is recommended tokeep the enzymes in a cold rack in the freezer, as this helps to stabilize the storage temperature.
Suboptimal reaction conditions
If there are no issues with digesting the control DNA, there may be something else wrong with yourreaction setup.
Solution check list
- ☑ Verify that you are using therecommended reaction buffer, including anyadditivesspecified in the product support material. Avoid improper buffers by using FastDigest restriction enzymes, which all work in the same buffer
- ☑ Make sure you are usingmolecular biology–grade water.
- ☑ Ensure that therestriction enzyme is the final component added to the reaction mix, and that the finalglycerol content is below 5%, so that the enzyme volume represents less than one tenth of the total reaction volume.
- ☑ Double check the optimalreaction temperaturefor the enzymes being used, andcontrol for evaporation during incubationas an increased salt concentration in the buffer can inhibit enzyme performance.
- ☑ Optimal DNA concentration is between 20–100 ng/µLin the final reaction mixture.
- ☑ DNA substrate is free of contaminants or reaction componentslike SDS, EDTA, protein, salts, and ethanol.
Enzyme activity blocked by DNA methylation
Several endogenous methylases site-specifically methylateadenine (DAM) or cytosine (DCM) residuesand can affect enzyme activity (Figure 2).
For example, methylation by deoxyadenosine methylase (DAM methylation) occurs normally in E. coli at GATC sequences. This sequenceoverlaps with the recognition sites of some enzymes, likeBamHIandBclI. In this case,BamHIcuts the DNA in the presence orabsence of methylation, whileBclIcannot cleave methylated DNA.To overcome this restriction, you can transform your plasmidDNA into a dam-minus,dcm-minus strain, such as E. coli GM2163. These methylation minus strains do not interfere withmethylation (Figure 3).
Figure 3. Genotype of a methylation minus strain.
Note: ApaI restriction enzyme is sensitive to CpG methylation. DAM/DCM covers only E. coli methylation. If you are working with eukaryotic DNA, you may have issues with restriction of this DNA due to CpG methylation.
The structure of substrate DNA
To approach this issue, first check what type of sample you are dealing with: PCR fragments or plasmids.
For PCR fragments:
Incomplete or no digestion of PCR products may be due to theproximity of the recognition site to the end of the DNA fragment.Some restriction enzymes requireadditional flankingbases for efficient DNA binding and cleavage (Figure 4).
Figure 4. Cut PCR products close to the DNA template end.Restriction site is usually at the 5′ end of the PCR primer.
Because recognition sites are often introduced at the ends of PCR fragments and/or primers, it is important to understand how many bases flanking a site are needed for optimal cleavage.
Enzyme suppliers often provide tables that illustratehow many bases from the end of a recognition site should be present for optimal activity.
For example, PasI can cleave DNA even if the recognition site is at the very end of the fragment, while PaeI requires at least 5 additional bases for optimal digestion (Figure 5).
Figure 5. Relative cleavage efficiencies for PaeI, PagI, and PasI as a function of the number of residues flanking their recognition sites.
For plasmid DNA:
If you are trying to perform a double digest with two enzymes in the multiple cloning site, efficient cleavage may be difficult if the two recognition sites are too close together. One enzyme may physically block access of the second enzyme to its respective site.
Inefficient cleavage is also related to the previously described proximity of the recognition site to DNA ends. After one enzyme cuts, there may not be enough bases flanking the second site for the second enzyme to bind and effect cleavage.
Due to the reasons above,consider doing a sequential digestion. Before you set up the reaction, determine which enzyme is more effective at cutting close to the end of a DNA fragment, and use that enzyme second.
For example, XbaI and SalI are next to each other in the pUC19 multiple cloning site (MCS) (Figure 6). If you cut with XbaI first, SalI would only cut with up to 20% efficiency.
Figure 6. pUC19 MCS.
However, if you cut with SalI first, XbaI can cut near the end of the strand, with up to 100% efficiency. For this reason, you should perform sequential reactions and digest with SalI followed by XbaI to prepare the plasmid for cloning (Figure 7).
Figure 7. Cleavage efficiency helps to determine the best order for sequential digests.
Insufficient incubation time
If your probable cause is insufficient incubation time, you should gradually increaseincubation time. Longer incubation times are often used to allow a reaction completion with fewer units of enzyme.
Enzyme concentration is low
In general, it is recommended to use3 to 5 units of enzyme permicrogramof DNA.Consultwith your supplier and product support materials to obtainrecommended enzyme concentration.If digestingsupercoiled DNA, increase enzyme units in the reaction.
Template is contaminated with inhibitors/PCR reaction components
Dealing with contaminated templates is common. Usespin column or PCR clean up kitto remove contaminants.Thevolume of DNA cannot exceed 25%of the total volume of the digestion reaction.
Unexpected cleavage pattern
Star activity and other related cause
- Star activity (off-target cleavage)
- High glycerol concentration (greater than 5% in the final reaction)
- High enzyme:DNA ratio, or overdigestion
- High pH or low ionic strength
- Presence of organic solvents such as DMSO or ethanol
- Use of a divalent cation other than magnesium in the reaction mix
- Inclusion of other non-optimal buffer conditions
- Prolonged incubation, such as overnight digestion
If you’re experiencing star activity or any of the causes above, here are all the key factors/areas you should check:
Choose an enzyme supplier that has addressed star activity as follows:
- ☑ Optimizes their enzymes and buffersto minimize star activity,
- ☑ Offersisoschizomerswith low or no intrinsic star activity, and
- ☑ Offers engineered or modified enzymes to eliminate star activity.
- ☑ Follow the recommendationsprovided with each enzyme for optimal activity including the use of thecorrect buffer, enzyme amount, and reaction time for the enzyme.
Most enzymes will not exhibit star activity when used under recommended conditions in optimal buffers. However, under suboptimal or extreme conditions, star activity may occur. Here’s a quick guide on How to Recognize Star Activity.
For example, incomplete digestion results in additional bands above the expected bands on a gel. These bands disappearwhen the incubation time or amount of enzyme is increased, as seen when comparing sample in lanes 2 and 3 tothe completely digested sample in lane 4.
Star activity, as seen in lanes 5 and 6, results in additional bands belowthe smallest expected size. These bands will generally become more intense with increasing enzyme dose ortime, while the expected bands become less intense (Figure 8).
Figure 8. Banding patterns for incomplete and complete digestion and star activity.
Most commercial enzymes are formulated with glycerol for stability and to prevent freezing at –20°C.
Gel-shift effect
Gel-shift is the result of another enzyme attribute and canresult in an unexpected banding pattern when viewing digested samples on a gel.It is typically more apparent when high enzyme doses are used and can have minor or significant impact on visualizing samples (Figure 9A).
A second method to reduce gel-shift is to add SDS into the loading buffer prior to loading on the gel.These methods will denature the enzyme, releasing it from the DNA fragment (Figure 9B).
Figure 9. Panel A: Gel shift effect. Panel B: Gel shift effect +/- SOS in the loading buffer.
Contamination/unexpected recognition sites
The restriction enzyme tube or reaction buffer tube may be contaminated with a second enzyme. This can happen where the same reaction buffer is used for multiple different enzymes.
- Try afresh tube of enzyme or reaction buffer.
- Check for contamination byusing afresh DNA preparation.
In rare cases, it may be possible that there are unexpected recognition sites in the substrate DNA.
You can check formutationsthat may have been introduced during PCR amplification. There is also potential to generatenew restriction sitesafter ligation of DNA fragments.
For example, some restriction enzymes have degenerate recognition sites. For example, XmiI cuts at GTMKAC, where M is either A or C, and K is either G or T. Make sure to check your substrate sequence for all potential sites (Figure 10).
Figure 10. Some restriction sites are degenerated and may result in unexpected banding patterns.
