Types Of Restriction Enzymes

Self-assembly cloning: a rapid construction method for recombinant molecules ...

Enzyme-free cloning (EFC) can rapidly produce an in-frame fusion gene with multiple fragments. To practically apply EFC, we investigated the extent and sequence of complementary staggered overhangs necessary to direct self-assembly of multiple fragments as well as a size limitation of the constructed DNA molecule. Six-base pair overhangs with 50% GC content were sufficient to direct self-assembly. A functional plasmid that exceeded 10 kb, which includes an in-frame fusion domain, was efficiently constructed from four PCR fragments in one step by our improved method.

Traditional cloning procedures based on restriction enzymes and DNA ligase are inefficient when constructing recombinant molecules with multiple fragments. Another limitation is the dependence upon restriction enzymes; a restriction site in the multicloning site of a plasmid often cannot be used when the insert contains a site for the same enzyme. This problem can be overcome by subcloning, but this is a time-consuming procedure. Enzyme-free cloning (EFC) ( 1 - 3 ) is a good alternative for producing in-frame fusion genes from multiple fragments, because it is simple, versatile, and cost-effective.

In this study, we modified the original EFC procedure ( 1 ) to clone multiple fragments in a single step (Supplementary Figure S1). Briefly, this was accomplished by performing two PCRs (one with the tailed forward primer and the untailed reverse primer, and the other with the untailed forward primer and the tailed reverse primer; Supplementary Table S1) to create the insert fragment. These two PCR products were treated with Dpn I to completely digest the template plasmid ( 4 ), mixed, denatured, and reannealed to create fragments having single-stranded overhangs.

Using the same procedure, vector fragments with staggered overhangs complementary to those of the insert were generated. A portion of the reactions for the inserts and the vector were mixed at room temperature, allowed to self-assemble, and used to transform competent cells. Our modified method depends upon Dpn I to digest template molecules after PCR, so it does not qualify as an EFC method; it is similar to EFC for self-assembly of multiple fragments, so we decided to call it self-assembly (SA) cloning.

We first identified the minimum length and optimal GC content necessary for the overhangs with one insert and one vector.

Restriction Enzymes (Molecular Biology)

Restriction enzyme refers here to the bacterial type II restriction endonucleases— one type of a general class of enzymes that function in restriction-modification systems (see Restriction- Modification Systems for other types of restriction enzymes). Type II restriction enzymes are ubiquitous and simple in composition. They are used extensively in recombinant DNA work and, in fact, made the technology possible. The restriction enzymes are Mg -requiring, homodimeric proteins that cleave at defined phosphodiester bonds within or adjacent to specific sequences of 4-8 base pairs (the "restriction site") of double-stranded DNA. The phosphodiester bond cleavage of each DNA strand produces DNA fragments with 5′ phosphate and 3′ hydroxyl termini. The scission of both strands results in either staggered-end or blunt-end double-strand DNA fragments (see Staggered Cut). Depending on the endonuclease used, the termini of the duplex DNA fragments may be completely duplex (blunt) or have protruding 5′ or 3′ single strands (otherwise known as "staggered", "sticky", or cohesive ends). Almost all type II restriction enzymes cut at DNA sequences that are palindromic, that is, have a dyad axis of symmetry, so that their complementary strand has the same sequence. Examples are the target sites for EcoRI (GAATTC) and FinII (CCGG). Some restriction enzymes classified as type II are monomeric enzymes and cut at defined distances outside specific asymmetric recognition sequences.

Approximately 2600 different restriction enzymes are known , with over 230 cleavage specificities (1). Bacteria synthesize minute amounts of the enzymes to serve their biological function (see Restriction-Modification Systems), but the overexpression of cloned restriction enzyme genes substantially facilitates their preparation and purification. The cognate modification methyltransferase partner of the restriction enzyme prevents endonucleolytic cleavage by methylating specific adenosine or cytosine nucleotides in the recognition sequence (see Methyltransferase, DNA), resulting in a sequence that is refractory to endonuclease hydrolysis.

Under optimized reaction conditions and presumably in vivo, restriction enzymes are highly selective for their canonical restriction sites. Changes in restriction enzyme buffer conditions, however, such as pH, ionic strength, divalent metal ion substitutions , or the addition of organic solvents, can induce inaccurate cutting (star activity). Different restriction enzymes that recognize and cut at the same position in identical recognition sites are referred to as isoschizomers. For example, the EcoRI and RsrI restriction enzymes both recognize and cut between the G and A of the canonical sequence, GAATTC. Other restriction enzymes, termed neoschizomers, recognize identical DNA sequences but cut at different positions within the sequence. For instance, Asp718I (GGTACC) and KpnI (GGTACC) cleave between the two Gs and between the two Cs, respectively.

Restriction endonucleases

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