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323.4. Cloning and expression of cyanamide hydratase (cah) marker gene
3.2.3.4.1. Site-directed mutagenesis in cah gene
The protocol provided by the manufacturer of Unique Site Elimination (USE)
mutagenesis kit (Pharmacia Biotech, Inc.. Piscataway, NJ. USA) was used to conduct
the site-directed mutagenesis on a recombinant plasmid, pUC8 which contained the
rah
gene devoid of a PvuI site. One target mutagenic primer and two selection
primers were employed for each desired mutation. In
rah
gene the position 81 and 84
with HindIII site encodes lysine and leucine respectively (Fig. 21). To remove the
HindIII site in
cah
gene without any change in lysine or leucine, the target mutagenic
primers, 5' GAC TCC CTG GGA
AAA
CTT GGT GAT 3' and 5' GAC TCC CTG
GGA AAG
CTC
GGT GAT 3' respectively, were used. In the primers, the underlined
codons replaced the original AAG and
C7T
codons for lysine and leucine respectively
in the
cah
gene. Designed to introduce a mutation into each of the two PvuI sites at
positions 387 and 1283 in the pUC8, the selection primers had the nucleotide
sequences 5' GTT GGG AAG GG C AAT TGG TGC GGG CCT C 3' and 5' CTT
CGG TCC TCC AAT TGT TGT CAG AAG 3'. respectively. The recognition
sequence CGATCG of PvuI site was changed to the underlined sequence in both of
the selection primers.
The reaction mixture contained, in a final volume of 30 p1, 0.025 pmol of
plasmid template and 1.25 pmol of each phosphorylated primer. After completion of
DNA synthesis and ligation, the reaction mixture was treated with 10 units of PvuI
restriction enzyme and then used to transform competent cells of a repair-defective
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