Figure 1.
Mechanisms of EMP and RF cloning.
Mechanistic details of EMP and RF cloning are compared. (A) EMP cloning involves two PCR reactions. In the 1st EMP PCR a forward primer F1 and a reverse primer R1 with overhang exponentially amplify the insert of interest. In the 2nd EMP PCR reaction the purified product is used as a megaprimer to exponentially amplify the target plasmid together with a forward primer F1 and reverse primer R2. (B) RF cloning also includes two PCR reactions. In the 1st RF PCR two overhang containing primers F1 and R1 exponentially amplify the insert of interest. In the 2nd RF PCR the purified product is used as a megaprimer to linearly amplify the target plasmid. Experimental procedures are described in detail in Materials and Methods and Tables S1 and S2.
Figure 2.
Analogy between PCR-based cloning techniques.
QuikChange and inverse PCR allow insertion of up to 50 bp, whereas RF and EMP cloning can accommodate inserts of up to 5 kb. QuikChange and RF cloning use linear amplification to obtain their product. This causes few PCR errors since potential mutations cannot be inherited in the next PCR cycle but also causes low product amounts. Inverse PCR and EMP cloning utilize exponential amplification to obtain high product amounts resulting in intensive, easily observable product bands, and large colony numbers.
Figure 3.
Time scale of a complete EMP cloning experiment.
The time scale of a typical EMP cloning experiment with a 1 kb insert and a 5 kb template vector is shown. Boxes indicate separate subroutines of the experiment. Triangles indicate a PCR product analysis and purification step.
Figure 4.
Cloning efficiency of EMP and RF cloning.
Cloning efficiency of EMP and RF cloning are compared. (A) Agarose gel band intensities of 2nd EMP and 2nd RF PCR reactions of 10 test cases are compared (insert length in parentheses). Complete agarose gels are shown in Figure S1. (B) The number of colonies obtained with EMP and RF cloning are compared (notice that the axis is logarithmic). (C) The cloning efficiency, depicted in number of correct clones of 10 analyzed, is compared for EMP and RF cloning. Agarose gels of restriction digests are shown in Figure S2.
Table 1.
Plasmid length, insert length and cloning efficiency of the 10 test cases.
Figure 5.
Applications of EMP cloning: One-step EMP.
EMP cloning allows for megaprimer production and insertion in a single PCR reaction. (A) In a one-step EMP PCR reaction limiting amounts of the overhang containing primer R1 are used to reach primer depletion in the first cycles. The resulting small amount of megaprimer generates a starting population of product template for the exponential amplification through forward primer F1 and reverse primer R2. (B) Product bands of one-step EMP are compared. 20 nM of primer R1 results in the most intense product band at ∼8 kb.
Figure 6.
Applications of EMP cloning: Multi-insert EMP.
EMP cloning allows for insertion of several megaprimers at once. (A) In a multi-insert EMP megaprimer 3′ overhangs are designed such that they bind in nested manner. The terminal insert (insert 1) then binds to the plasmid backbone 3′ of the insertion site. In an exponential amplification reaction forward primer F1 binds to the beginning insert (insert 3) ensuring that all inserts are getting amplified. Reverse primer R2 binds 5′ of the insertion site. (B) A test digest of nine colonies obtained by multi-insert EMP of three inserts shows one correct clone (number 4).