Troubleshooting Common Issues with Restriction Digestion Reactions - IL (2024)

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 issueProbable causes
Incomplete or no digestion
  1. Inactive restriction enzyme
  2. Suboptimal reaction conditions
  3. Enzyme activity blocked by DNA methylation
  4. Substrate DNA structure
  5. Insufficient incubation time
  6. Low enzyme concentration
  7. Contaminated template
Unexpected cleavage pattern
  1. Gel-shift Effect
  2. Star activity and other related causes
  3. Contamination and unexpected recognition sites

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.

Troubleshooting Common Issues with Restriction Digestion Reactions - IL (2024)

FAQs

Why is my restriction digestion not working? ›

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 a fresh tube of enzyme or reaction buffer. Check for contamination by using a fresh DNA preparation.

What are the factors affecting restriction digestion? ›

Temperature - Most restriction enzymes work most efficiently at 37°C, but there are a few exceptions. Some work best at higher temperatures ranging from 50°C to 65°C, while a few cut best at lower temperatures of about 25°C. Buffer systems - Most restriction enzymes exhibit maximum activity in the pH range of 7.0–8.0.

How to check if restriction digestion has happened? ›

Restriction Digest Troubleshooting

Run a gel: After you cut your DNA (both insert and backbone), you should check the size on a gel. Run a DNA agarose gel with your digested plasmid alongside a lane of the uncut plasmid. Your uncut plasmid should appear to run smaller due to the supercoiled nature of uncut plasmid.

How to stop restriction digestion reaction? ›

If further manipulations of the digested DNA are required, heat inactivation (raising the temperature to 65 or 80°C for 20 minutes) is the simplest method of stopping a reaction.

How to improve restriction enzyme digestion? ›

  1. Use no more than the recommended enzyme amount (e.g., 10 units of enzyme per microgram of DNA). ...
  2. Avoid prolonged incubation of the digestion reaction.
  3. Use the recommended reaction buffer. ...
  4. Verify that no more than 5% glycerol is in the reaction.

Why my digestion is not working properly? ›

Digestion problems may be a result of stress or digestive conditions. Mild digestive problems may respond well to at-home treatments, such as adding or removing certain foods from the diet, exercise, and keeping a food diary. However, more serious issues may require medical attention.

How to fix incomplete digestion? ›

Diet and lifestyle changes can make a big difference:
  1. Cut back on fatty foods.
  2. Avoid fizzy drinks.
  3. Eat and drink slowly.
  4. Quit smoking.
  5. Don't chew gum.
  6. Exercise more.
  7. Avoid foods that cause gas.
  8. Avoid sweeteners that cause gas such as fructose and sorbitol.
Mar 25, 2023

How can you confirm the success of a restriction digestion reaction? ›

Control DNA (DNA with multiple known sites for the enzyme, e.g. lambda or adenovirus-2 DNA) with restriction enzyme to test enzyme viability. If the control DNA is cleaved and the experimental DNA resists cleavage, the two DNAs can be mixed to determine if an inhibitor is present in the experimental sample.

How long does restriction digest take? ›

Incubate tube at appropriate temperature (usually 37 °C) for 1 hour. Always follow the manufacturer's instructions. Depending on the application and the amount of DNA in the reaction, incubation time can range from 45 mins to overnight. For diagnostic digests, 1-2 hours is often sufficient.

How do you know if you lack digestive enzymes? ›

Symptoms of Digestive Enzyme Insufficiency

Bloating. Diarrhea. Gas. Oily stools (bowel movements)

What to do after restriction digestion? ›

Stop the digestion by heat inactivation (65 °C for 15 minutes) or addition of 10 mM final concentration EDTA. Recover the DNA using a purification kit: re-suspend and dilute the DNA to 1 µg/µL. Prepare second digestion according to step 1.

Why would restriction digestion fail? ›

The most common reason a restriction digestion fails is the presence of a contaminant in your experimental DNA that's inhibiting the restriction enzyme. Contaminants include phenol, ethanol, chloroform, excess salts, detergents, or EDTA.

What is a typical restriction digest reaction? ›

The components of a typical restriction digestion reaction include the DNA template, the restriction enzyme of choice, a buffer and sometimes BSA protein. The reaction is incubated at a specific temperature required for optimal activity of the restriction enzyme and terminated by heat.

How to get rid of restriction enzymes? ›

Restriction enzymes are commonly inactivated by a heat treatment after digestion is complete.

Why aren't my digestive enzymes working? ›

The environment in the digestive system requires a delicate balance. Replacement digestive enzymes may not work well if the environment in your small intestine is too acidic due to a lack of bicarbonate. Another issue can be that you're not taking the right dose or ratio of enzymes.

Why is my body refusing to digest food? ›

A damaged vagus nerve can't send signals normally to your stomach muscles. This may cause food to remain in your stomach longer, rather than move into your small intestine to be digested. The vagus nerve and its branches can be damaged by diseases, such as diabetes, or by surgery to the stomach or small intestine.

Why is my body not fully digesting food? ›

Sometimes, you may see undigested food fragments in stool. This usually is high-fiber vegetable matter, which usually isn't broken down and absorbed in your digestive tract. At times, undigested food is a sign of poor chewing and fast eating. Make sure that you chew your food well.

Why would a restriction enzyme not cut? ›

If an inhibitor (often salt, EDTA or phenol) is present, the control DNA will not cut after mixing. Additional troubleshooting help is also available. DNA methylation is also an important element of a restriction digest.

Top Articles
Latest Posts
Recommended Articles
Article information

Author: Rev. Leonie Wyman

Last Updated:

Views: 5797

Rating: 4.9 / 5 (79 voted)

Reviews: 94% of readers found this page helpful

Author information

Name: Rev. Leonie Wyman

Birthday: 1993-07-01

Address: Suite 763 6272 Lang Bypass, New Xochitlport, VT 72704-3308

Phone: +22014484519944

Job: Banking Officer

Hobby: Sailing, Gaming, Basketball, Calligraphy, Mycology, Astronomy, Juggling

Introduction: My name is Rev. Leonie Wyman, I am a colorful, tasty, splendid, fair, witty, gorgeous, splendid person who loves writing and wants to share my knowledge and understanding with you.