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

Schematic representation of a cloning strategy using a Body Double ENase.

<p>The use of a Type IIP ENase is not feasible for creating the desired overhang at the end of a PCR fragment due to the presence of an internal restriction site of the Type IIP ENase in the PCR fragment. <b>A.</b>) Digestion by the Type IIP restriction endonuclease cleaves the PCR fragment into two parts. Alternative strategy needs to be exploited. <b>B.</b>) Digestion by a Body Double Type IIS restriction ENase creates the appropriate overhangs on the PCR fragment identical to the overhangs that would be generated by the Type IIP ENase. The PCR fragment remains intact. REP A and B: Type IIP restriction enzymes, their recognition sites are represented by red and blue boxes, respectively. 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 sites corresponding to a Type IIP overhang are represented by an orange box. The flanking bases with arbitrary sequence needed for efficient enzymatic digestion of the PCR fragment are marked by light grey.</p

List of ENases usable as Body Doubles.

<p>Commercially available enzymes suitable for being Body Doubles of a Type IIP ENase are grouped according the type of the generated overhangs. ENases of which recognition and cleavage sites need more than 18 bases to be accommodated in a primer are not listed. Isoschizomers are listed in the same row, separated by commas.</p>1<p>) No data about religation efficiency.</p>2<p>) No data about cutting predictability.</p>3<p>) Type IIP ENase with interrupted recognition site.</p

The primers used in this publication that contain Body Double restriction enzyme sites.

<p>The recognition sites of the enzymes are in <i>italic</i>; the overhangs generated are in CAPITALS. The cutting position is marked by a bold apostrophe. <b>Bold</b> and <u>underline</u> marks the bases which were altered in order to change the recognition sequence of the Type IIP ENase used.</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

Ligation of NcoI overhangs generated by NcoI and its Body Double reconstitutes the NcoI recognition site.

<p><b>A.</b>) Sequence of NcoI recognition and cleavage site <b>B.</b>) Sequence of BsmBI recognition and cleavage site, the latter corresponding to an NcoI overhang <b>C.</b>) Ligation of the NcoI overhangs generated by different restriction enzymes reconstitutes the NcoI site. REP: Type IIP restriction enzymes, their recognition sites are represented by red 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 sites correspond to a Type IIP overhang are represented by the same orange box.</p

Schematic representation of a cloning strategy using a Body Double ENase when the Type IIP recognition sites in the vector are not reconstituted by the ligation at <i>A.)</i> one of the ends or <i>B.)</i> both ends of the insert.

<p>Digestion with a Body Double Type IIS restriction endonuclease creates the appropriate overhangs on the PCR fragment that are identical to the overhangs generated by the Type IIP ENase. <b>A.</b>) Only one of the REP restriction sites is reconstituted by the ligation at one end of the insert, and thus a unique REP site is formed in the vector. <b>B.</b>) The REP restriction sites are not reconstituted by the ligation at either ends of the insert. No REP site remains in the vector. REP: Type IIP restriction enzyme, its recognition site is 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 green boxes. The flanking bases needed for efficient enzymatic digestion of the PCR fragment are marked in light grey.</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