TABLE 1.
Primers used in this study
| Primer | Sequence 5′→3′ | Amplification target |
|---|---|---|
| Northern blot probe PCR | ||
| Sta2 For | GCGTATGTTCAATTTGTCG | 1,140-bp product from lst of N. gonorrhoeae and N. meningitidis |
| Sta3 Rev | CGTCAAATGTCAAAATCGG | |
| Real-time PCR | ||
| LstRTF | AAACCCGCATACGAGGTATGA | 100-bp product from lst of N. gonorrhoeae and N. meningitidis |
| LstRTR | AAGCCGGTTTCAATGCGTAA | |
| 16S RT F | GCGTGGGTAGCAAACAGGAT | 100-bp product from rrs gene of N. gonorrhoeae and N. meningitidis |
| 16S RT R | CGCGTTAGCTACGCTACCAAG | |
| Cloninga | ||
| FusBam F1 | CGCTGGATCCGACATCAATATCGG | 496 bp of N. gonorrhoeae 5′lst including RBS sequence and 24 bp of lst including ATG |
| FusBam R1 | CAAAGGATCCTTTTTCAAGCCC | 586 bp of N. meningitidis 5′lst including RBS sequence and 24 bp of lst including ATG |
| Primer extension | ||
| PE1 | CGCATTCCTTTCCCCCTGATTTAC | 3′ region of primers anneals 78, 16, and 24 bp downstream of lst ATG, respectively |
| PE2 | CACAACACGGTCAAACAAGC | |
| PE3 | CAATCAGGCACAACACGGTC | |
| RPA primers | ||
| PA1 | GATCGAGCTCGTTCGATCTTGGCGTGTTTG | 292 bp of N. gonorrhoeae 5′lst excluding the RBS sequence; SacI site of PA1 is denoted by double underline |
| PA2 | GATCGGATCCCTCCATTCCGACAAATTGAAC | |
| PA3 | AATTAACCCTCACTAAAGGG | 397-bp product from pSKII including the cloned 292-bp N. gonorrhoeae 5′lst insert |
| PA4 | GTAATACGACTCACTATAGGG | |
| RT-PCR: Fig. 7 | ||
| P1 | CGGGATCCGGCTTTCCCGCGTTTGCCGG | 5′ region of the primer anneals upstream of CREE; 282 bp upstream of lst ATG |
| P2 | CGGGATCCCGCCTTGTGCCTGATGTGCG | 5′ region of the primer anneals upstream of CREE; 252 bp upstream of lst ATG |
| P3 | CGGGATCCTTTCGGTAAAATTGATTTTA | 5′ region of the primer anneals upstream of CREE; 222 bp upstream of lst ATG |
| P4 | CGGGATCCAACTGTCGGAATATCTGCTA | 5′ region of the primer anneals downstream of CREE; 91 bp upstream of lst ATG |
| P5 | CGGGATCCTTTTTCCGTCCCGGGACAC | 5′ region of the primer anneals downstream of CREE; 61 bp upstream of lst ATG |
| P6 | CGGGATCCACACTCGGGGCGTATGTTCA | 5′ region of the primer anneals downstream of CREE; 41 bp upstream of lst ATG |
| P7 | CGGGATCCAGGGATATGGGCTTGAAAAAG | 5′ region of the primer anneals downstream of CREE; 6 bp upstream of lst ATG |
| Crev | CTCCGCCATCGTCGGAAT | Common reverse primer used in conjunction with primers P1 to P7 and the 3′ region of the primer anneals 372 bp downstream of lst ATG |
| RT-PCR: Fig. 8 | ||
| IP1 | TTATTCTCTCTTGTAGGTTGG | Generates a 212-bp product of 5′icd upstream region; P4 and Crev primers used in Fig. 7 were used in Fig. 8 |
| IP2 | TGCCGCCGCACATCAGGCACA |
a
Primers FusBam F1 and FusBam R1 were used to include regions of the N. gonorrhoeae or N. meningitidis 5′lst region including the RBS and 24 bp of the lst gene including ATG to create translational lacZ fusions in pLES94. The BamHI restriction sites are underlined with single lines.