TABLE 1.
Primer sequences used in site-directed mutagenesis
| Mutant | Mutagenic primera | Orienta-tionb | Primer Ic | Primer IId |
|---|---|---|---|---|
| Metal-binding site | ||||
| D97A | TACGGTGACAgCAATGGTAAC | A | R | U |
| D108A | TTTCGAGGTAgCGCCGTGGAA | A | R | U |
| H171L | TATTGCGGACtCGGTATTGGT | S | U | R |
| E204V | TTCACCATCGtGCCAATGGTC | S | U | R |
| E235V | GCACAATATGtGCATACTAAT | S | U | R |
| Hydrophobic pocket | ||||
| C59S | GTTTCTGCCaGCCTCGGCTAT | S | R | U |
| C70S | AATAGAGATGCtAACGGATTT | A | U | R |
| Y62F | ATAGCCGTGAaAGCCGAGGCA | A | U | R |
| Y65F | GGATTTCGGAaAGCCGTGATA | A | U | R |
| F177L | GGTCGCGGCTTgCATGAAGAA | S | R | U |
| W221L | AAAGATGGCTtGACGGTAAAA | S | R | U |
a
Nucleotides that create the desired mutation are presented in lowercase letters.
b
Orientation of the mutagenic primer corresponding to the sense (S) or antisense (A) strand of the E. coli MAP gene.
c
Primers that are paired with the mutagenic primer for PCR for generation of the megaprimer.
d
Primers that are paired with the megaprimer for the second PCR. U, universal primer (5′-GTAAAACGACGGCCAGC-3′); R, reverse primer (5′-GGAAACAGCTATGACCATG-3′).