Restriction Enzymes for Cloning and DNA Analysis
Restriction enzymes (restriction endonucleases) are bacterial proteins that recognize specific short DNA sequences of 4-8 bp and cleave double-stranded DNA at or near those sites, generating defined sticky or blunt ends for use in restriction cloning, diagnostic digestion, restriction fragment length polymorphism (RFLP) analysis, Golden Gate assembly, and DNA mapping.
MBP supplies restriction enzymes for research labs across the United States, Canada, and internationally, with US order processing in Houston, Texas, and same-day dispatch on in-stock items. Request a quote by contacting customerservice@mbpinc.net.
Restriction Enzymes
Showing 1 to 3 of 3 results
What Are Restriction Enzymes?
Restriction enzymes are endonucleases originally isolated from bacteria, where they function as a primitive immune defense against foreign DNA (such as bacteriophages) by cleaving at specific recognition sequences. Over 3,500 Type II restriction enzymes have been characterized, recognizing palindromic or asymmetric sequences of 4-8 base pairs and generating either 5' overhangs (sticky ends), 3' overhangs, or blunt ends. The systematic naming convention reflects enzyme origin: EcoRI comes from Escherichia coli strain R; HindIII from Haemophilus influenzae strain Rd. Restriction enzymes are grouped into Types I, II, III, and IV based on cofactor requirements, cleavage site position, and structural complexity; Type II enzymes are almost exclusively used in research applications due to their defined cleavage positions. Choose restriction enzymes based on the recognition sequence present in your target, the end type required (sticky vs. blunt), and whether Golden Gate Assembly or conventional cloning is planned.
What you will find:
-
Enterokinase Cleavage Enzyme: A specific mammalian-produced protease for precise affinity tag removal from recombinant fusion proteins.
-
DpnI: A specialized enzyme targeting methylated GATC sequences, ideal for template digestion in site-directed mutagenesis.
-
Tn5 Transposase for Tagmentation: A high-efficiency enzyme for simultaneous DNA fragmentation and adapter ligation in NGS library construction.
How to Choose Restriction Enzymes
Type IIP vs. Type IIS
Type IIP enzymes (EcoRI, HindIII, BamHI, NcoI, NheI) recognize palindromic sequences and cut within or immediately adjacent to the site, making them the standard choice for conventional restriction cloning using compatible cohesive ends. Type IIS enzymes (BsaI, BsmBI/Esp3I, SapI) cut at a fixed distance outside their recognition sequence, generating custom 4-nt overhangs that can be designed to create any desired sequence at the assembly junction -- the basis of Golden Gate Assembly, which allows ordered multi-fragment cloning in a single tube.
Recognition sequence frequency
4-cutter enzymes (MboI, AluI, TaqI) cut on average every 256 bp and are used for genome-wide restriction mapping or chromatin fragmentation. 6-cutters (EcoRI, HindIII) cut every ~4,000 bp on average and are standard for plasmid cloning. 8-cutters (NotI, PacI, AscI) cut rarely (every ~65,000 bp) and are used for mapping of large genomic regions or release of large inserts from BAC or YAC vectors.
Star activity sensitivity
Restriction enzymes can exhibit relaxed sequence specificity (star activity) at suboptimal conditions: excess glycerol (>5%), non-standard buffers, extended digestion times, or non-standard divalent cations. High-fidelity (HF) enzyme variants reduce star activity by more than 1000-fold and are recommended for long digestion times or when high specificity is critical for downstream cloning.
Isoschizomers and compatible ends
Isoschizomers recognize the same sequence as a reference enzyme; neoschizomers recognize the same sequence but cut at a different position. BamHI (GGATCC, 5' overhang G) is compatible with BglII (AGATCT) overhangs and with MboI and Sau3AI overhangs -- useful for directional cloning strategies without re-engineering sites. Check isoschizomer tables before designing multi-enzyme cloning strategies.
Buffer compatibility
Most suppliers have rationalized their enzyme buffers into 2-4 universal buffer systems (e.g., CutSmart, Buffer 3.1, FastDigest buffers) that support the majority of enzymes, enabling double digestion without buffer exchange. Verify buffer compatibility before combining two restriction enzymes in a single tube.
Specifications Context
Restriction enzyme activity is defined as the amount of enzyme required to digest 1 microgram of lambda phage DNA (or substrate specified by the manufacturer) to completion in 1 hour at 37 degrees C in 50 microliters total volume. FastDigest enzyme formats complete digestion in 5-15 minutes at 37 degrees C, using a universal buffer. Heat inactivation is possible for most enzymes at 65-80 degrees C for 20 minutes, though some enzymes (NheI, SpeI, BstEII) are not heat-inactivatable and require column cleanup before downstream ligation. Over 3,500 Type II restriction enzymes are characterized in the REBASE database; commercially available catalogues cover approximately 200-300 common enzymes. For ligating restriction-digested fragments, see DNA-modifying enzymes for T4 DNA Ligase and alkaline phosphatase.
Refine your genomic editing—contact the MBP team today to request a quote for our high-fidelity enzymes.
FAQ
