( resistant. The small amount of inser...)Expression and Purification of Recombinant Proteins That Have Native Amino,,,Acid Sequence

resistant. The small amount of insert DNA required for LIC cloning can be obtained from only 10 to 15 cycles of PCR amplification with a high-fidelity enzyme, thus minimizing sequence errors generated by PCR. (For Pfu DNA polymerase, 10 rounds of PCR amplification of a 1kb template results in approximately 99% of the PCR products being free of errors.)

No Constraints on Sequence of Insert DNA

The insert DNA is prepared for cloning by treatment with Pfu DNA polymerase in the presence of dATP. The 3 to 5'-exonuclease activity of Pfu DNA polymerase will remove nucleotides until it encounters the first adenine (figure 2), at which point the 5 to 3-polymerase activity of Pfu DNA polymerase will incorporate the supplied dATP into the sequence.

The PCR product of the pCAL-n-EK vector DNA contains an adenine residue following the C terminus of the insertspecific sequence, which provides a stopping point for the exonuclease activity of Pfu DNA polymerase (figure 2). The N terminus of the insert DNA sequence may not contain an adenine residue for several base pairs past the single-stranded complementary region to the pCALnEK vector, causing a single-stranded gap to form upon annealing insert to the pCALnEK vector. In order to test the effect of single-stranded gaps on the efficiency of LIC cloning, primers were constructed that would produce 17-nucleotide and 27-nucleotide gaps upon annealing the JNK PCR product to prepared pCAL-n-EK vector. (In most experimental cases, gaps will be less than 17 nucleotides long.) Of a 20l annealing reaction, 1 l was transformed into XL1Blue supercompetent cells, resulting in an average of 1250 cfu. Nine clones were sequenced, and eight were found to contain perfect sequence: the 17 and 27-nucleot
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