Site-directed mutagenesis is an essential tool for the study of structure-function relationships. A variety of methods have been developed for modifying specific amino acids at predetermined sites in proteins. We present here an improvement of the method based on homologous recombination described by Jones and Howard (1) which not only largely diminishes the background of non-mutated clones but also greatly simplifies the whole mutagenesis procedure. The method combines both polymerase chain reaction (PCR) technology and homologous recombination. Briefly, an entire circular plasmid is amplified by PCR using mutagenic primers with overlapping sequences (25-base overlap). The linear fragment thus obtained bears homologous ends. After separation from the circular template by electrophoresis on an agarose gel, the linear fragment is transformed into competent DH5a E.coli cells. Homologous recombination occurs in the bacteria and yields clones harbouring a mutated circular plasmid. We have applied this technique with success to a pUC19 plasmid containing the sequence coding for mature BDNF (brain-derived neurotrophic factor). The size of the plasmid is 3.1 kilobases (kb), and 100% of mutated clones were obtained. However when the same strategy was applied to a pUC19 plasmid containing the prepro-BDNF sequence (ppBDpUC3), which is 3.6 kb long, the efficiency of mutagenesis dropped to 20-25%. This decrease in efficiency was explained by the difficulty of separating linear from supercoiled DNA above a certain size. Since the transformation efficiency is much higher with supercoiled than with linear plasmid, the low amount of contaminating supercoiled template plasmid yields a high background of non-mutated plasmid after transformation. To circumvent this problem we decreased the amount of template plasmid in the PCR reaction. An appropriate ratio between linear mutated PCR product and circular wild-type template is thus obtained and allows direct transformation into bacteria. The primers BDXHOA 5'-CTC GAG GAC TGT GAC CGT CCC GCC AGA CAT GTC CAC TGC-3' and BDXHO1 5'-TGG CGG GAC GGT CAC AGT CCT CGA GAA AGT CCC GGT ATC C-3' were designed to create a silent XhoI restriction site in the mouse BDNF sequence at position 582-587 in the sequence available in GenBank under the accession number X55573. The NI -; Figure 1. Effect of decreasing amounts of template on PCR yield. Electrophoresis was performed on a 1% agarose gel stained with ethidium bromide at 0.5 jg/ml. M: k HindIIllEcoRI marker (Appligene). Lanes 1-3: 10 .l of PCR amplification performed respectively with 1 ng, 0.1 ng, 0.01 ng of the 3.6 kb-long ppBDpUC3 plasmid.mutagenic nucleotides are in italic bold characters and the overlapping regions are underlined. Amplification of full-length plasmid was performed in a 100 ,l incubation volume containing 1 x buffer (10 mM Tris-HCI pH 9; 50 mM KCI; 1.5 mM MgCl2; 0.1% Triton X-100; 0.2 mg/ml BSA or gelatin), 200 ,uM of each deoxynucleoside triphosphate, 1 FM of each primer and 2.5 U of Taq polymerase (Appligene) using a Per...
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