Restriction Enzymes (Restriction Endonucleases)

Restriction enzymes, also known as restriction endonucleases, are essential tools in molecular biology. These enzymes recognize and cut DNA at specific nucleotide sequences, enabling researchers to manipulate DNA with high precision. Originally discovered in bacteria as a defense mechanism against viruses, restriction enzymes are now indispensable in cloning, genome editing, forensic analysis, and diagnostics.

How Restriction Enzymes Work

Restriction enzymes identify short, specific DNA sequences (recognition sites), usually 4–8 base pairs long. Upon recognition, they cleave the DNA by hydrolyzing the phosphodiester bonds within or near these sequences. This cleavage generates DNA fragments, either with blunt ends or sticky ends:

1. Blunt-end cuts: Produce straight cuts through both DNA strands at the recognition site, yielding flat ends.

2. Sticky-end cuts: Generate overhanging single-stranded ends, which can easily anneal with complementary sequences.

To prevent self-cleavage, host bacteria use methylases to methylate their own DNA at the recognition sites, forming a restriction-modification system that distinguishes foreign DNA from self.

Types of Restriction Enzymes

Restriction enzymes are categorized based on their structure and cleavage patterns:

1. Type I: Large multi-subunit complexes that cut DNA far from their recognition sites; require ATP and S-adenosyl methionine. Example: EcoKI.

2. Type II: Most widely used in labs. Cut DNA at or near the recognition site. Do not require ATP. Examples: EcoRI, HindIII, BamHI.

3. Type III: Multi-subunit enzymes requiring two recognition sites and ATP. Cut DNA at a defined distance away. Examples: EcoP15, EcoP1.

Applications of Restriction Enzymes

1. Gene cloning and plasmid construction

2. DNA fingerprinting in forensics

3. Diagnostic assays for genetic disorders

4. Genome editing, including CRISPR applications

5. Library construction and genome mapping

Choosing the Right Enzyme

When selecting a restriction enzyme for DNA digestion, consider:

1. Recognition site: Ensure the enzyme targets the exact DNA sequence of interest.

2. End type: Choose between sticky or blunt ends depending on downstream applications.

3. Buffer compatibility: Especially important in multi-enzyme reactions.

4. Methylation sensitivity: Some enzymes can't cleave methylated DNA.

5. Star activity: Avoid enzymes prone to off-target cleavage under suboptimal conditions.

6. Temperature and reaction time: Follow recommended conditions for optimal activity.

Tips for Successful Digestion

1. Maintain glycerol concentration below 5% in the reaction mix.

2. Use the correct buffer with necessary cofactors (e.g., Mg2+).

3. Optimize incubation time to avoid incomplete digestion or star activity.

4. Store enzymes at recommended temperatures to preserve activity.

BioFargo Restriction Enzymes

At BioFargo, we provide a curated selection of high-performance restriction enzymes, rigorously tested for purity, specificity, and lot-to-lot consistency. Our restriction enzyme portfolio is marketed under the KleanAB brand, known for its superior quality and performance.

KleanAB™ Restriction Enzymes are designed to meet the needs of researchers in gene cloning, diagnostics, and high-throughput applications. Our catalog includes commonly used enzymes such as EcoRI, BamHI, HindIII, and unique enzymes for specialized needs.
We also offer:

1. Fast-digest enzymes for rapid results in under 15 minutes

2. Universal buffer systems for simplified reaction setup

3. High-stability formats for extended shelf life

Examples of specialized enzymes:

Explore our full enzyme catalog to find the ideal tool for your molecular biology applications.

Have questions or need help selecting an enzyme? Contact our technical team—we’re happy to help!