Restriction Enzyme Body Doubles and PCR Cloning: On the General Use of Type IIS Restriction Enzymes for Cloning

<div><p>The procedure described here allows the cloning of PCR fragments containing a recognition site of the restriction endonuclease (Type IIP) used for cloning in the sequence of the insert. A Type IIS endonuclease - a Body Double of the Type IIP enzyme - is used to generate the same protruding palindrome. Thus, the insert can be cloned to the Type IIP site of the vector without digesting the PCR product with the same Type IIP enzyme. We achieve this by incorporating the recognition site of a Type IIS restriction enzyme that cleaves the DNA outside of its recognition site in the PCR primer in such a way that the cutting positions straddle the desired overhang sequence. Digestion of the PCR product by the Body Double generates the required overhang. Hitherto the use of Type IIS restriction enzymes in cloning reactions has only been used for special applications, the approach presented here makes Type IIS enzymes as useful as Type IIP enzymes for general cloning purposes. To assist in finding Body Double enzymes, we summarised the available Type IIS enzymes which are potentially useful for Body Double cloning and created an online program (<a href="http://group.szbk.u-szeged.hu/welkergr/body_double/index.html" target="_blank">http://group.szbk.u-szeged.hu/welkergr/body_double/index.html</a>) for the selection of suitable Body Double enzymes and the design of the appropriate primers.</p></div

Cloning efficiency of constructs made by using BD (1–21) or Type IIP enzymes (C1–C20).

<p>The number of colonies containing the correct constructs was counted for each experiment and the percentage of those containing the correct constructs as a proportion of all tested clones was calculated. Experiments 1 to 21 were made using BD enzymes (blue, orange, yellow), and experiments C1 to C20 were made using Type IIP enzymes with background clearing (green). There was no possibility of decreasing the background in the experiments represented by orange columns. The constructs represented by yellow columns were made by inserting multiple inserts into the vector in a single ligation step. These experiments were not included in the calculation of the average BD efficiency. The efficiencies are 84,38% for the BD constructs and 83,80% for the Type IIP constructs. There is no data for samples 8 and 9.</p

Ligation of AgeI overhangs generated by AgeI and its Body Double does not reconstitute the AgeI recognition site.

<p><b>A.</b>) Sequence of AgeI recognition and cleavage site <b>B.</b>) Sequence of BsmBI recognition and cleavage site, the latter corresponding to an AgeI overhang <b>C.</b>) Ligation of the AgeI overhangs generated by different restriction enzymes does not reconstitute the AgeI site. REP: Type IIP restriction enzymes, their recognition sites are represented by orange boxes. The sequences between the cutting positions corresponding to the overhangs are indicated by altered coloring in the boxes. RES: Body Double Type IIS restriction endonuclease, its recognition site is represented by a green box, the sequences between its cleavage site correspond to a Type IIP overhang are represented by a yellow box. Arbitrary bases (N) are light grey, altered bases that destroy the AgeI recognition site are shown in black. V and B represent G,C or A and G,C,or T, respectively.</p

Efficiency of background clearing.

<p>1.8% Agarose gel showing clones with (1–10) and without (11–20) background clearing (case 19). The plasmid DNA purified from colonies digested with NdeI for the presence of insert DNA. Background clearing was carried out before transforming the ligation reactions to competent <i>E. coli</i> bacteria by digesting (clones 1 to10) or without digesting (clones 11 to 20) the ligation reactions with the appropriate Type IIP enzyme (AgeI) used for cloning. The acceptor vector contains an AgeI site while the correct product vector does not. Thus, AgeI digestion linearized the circular acceptor vectors that contain no inserts thereby decreasing the empty vector background. After background clearing (1 to 10) none of the colonies contains an empty vector. In contrast, without background clearing 9 out of the 10 colonies have empty vectors. Some of the constructs apparently contained more than one insert. (No.: Numbers of the tested colonies, BC: background clearing, D: test digestion (expected fragments after NdeI digestion: 842, 4410; if multiple inserts were ligated, the length of the 842 fragment increases by ∼100 for each additional insert), C: control plasmid (expected fragments after NdeI digestion: 743, 4410), L: DNA ladder). The red line represents the mobility of the 743 bp NdeI fragment of the control vector.</p

Layouts of generalized primers containing BD ENase sites useable to generate different type of overhangs and used in different orientations.

<p>These layouts are based on the outputs of the Body Double Finder online program. A primer containing the site of a RES generating <b>A.</b>) 4 nt, 5′ overhang <b>B.</b>) 2 nt, 5′ overhang and <b>C.</b>) 2 nt, 3′ overhang. To the left is the recognition sequence of BsaI, FauI and BseRI respectively (recognition site: green, purple and red letters; cleavage site: orange, blue and yellow letters respectively). Green, purple and red triangles mark where cutting occurs, and the lines show the overhangs generated. To the right are possible primer layouts. In the primers shown uppermost the RES removes its recognition site from the PCR fragment while in the other primer (below) the recognition site of the Body Double RES remains in the insert after digestion. In the first case the oligo primer must contain an oligonucleotide tail in order to facilitate the cleavage by the restriction enzyme at the very edge of the primer. Such an extra tail is not required in the second case since the cleavage site already flanks the recognition site. Increasing the distance between the recognition and cleavage sites results in longer primers, therefore in <b><i>C.</i></b><i>)</i> only one orientation of the Body Double RES site can be accommodated in a PCR primer of the suggested length.</p