TABLE 1.
Probe sequences used in the oligonucleotide biochip assaya
| Gene and probe | Oligonucleotide sequence | Tm (°C) |
|---|---|---|
| gyrA | ||
| Probe1 (Ser91; wild type) | 5′-NH2-(T)16-CGATTCCGCAGTTTA-3′ | 49.5 |
| Probe2 (Phe91; mutation) | 5′-NH2-(T)16-CGATTTCGCAGTTTA-3′ | 49.1 |
| Probe3 (Asp95; wild type) | 5′-NH2-(T)16-AGTTTACGACACCATC-3′ | 46.8 |
| Probe4 (Asn95; mutation) | 5′-NH2-(T)16-AGTTTACAACACCATCGT-3′ | 50.2 |
| Probe5 (Ala95; mutation) | 5′-NH2-(T)16-TTACGCCACCATCG-3′ | 49.3 |
| Probe6 (Gly95; mutation) | 5′-NH2-(T)16-TTACGGCACCATCG-3′ | 49.3 |
| parC | ||
| Probe7 (Asp86/Ser87; wild type) | 5′-NH2-(T)16-CACGGCGACAGTTCC-3′ | 49.2 |
| Probe8 (Asn86; mutation) | 5′-NH2-(T)16-GCACGGCAACAGTTCC-3′ | 51.3 |
| Probe9 (Arg87; mutation) | 5′-NH2-(T)16-CGACCGTTCCGCCT-3′ | 50.7 |
| Probe10 (Asn87; mutation) | 5′-NH2-(T)16-CGACAATTCCGCCTAT-3′ | 48.9 |
| Probe 11 (Ile 87; mutation) | 5′-NH2-(T)16-CGACATTTCCGCCTAT-3′ | 47.9 |
| Probe 12 (Arg 87; mutation) | 5′-NH2-(T)16-CGACAGGTCCGCCTA-3′ | 49.2 |
| Probe 13 (Glu91; wild type) | 5′-NH2-(T)16-CTATGAGGCGATGGTG-3′ | 52.4 |
| Probe 14 (Lys91; mutation) | 5′-NH2-(T)16-CCTATAAGGCGATGGTG-3′ | 54.6 |
| Probe 15 (Gly91; mutation) | 5′-NH2-(T)16-CCTATGGGGCGATG-3′ | 52.8 |
| Probe 16 (Ala 91; mutation) | 5′-NH2-(T)16-CCTATGCGGCGATG-3′ | 52.8 |
| Probe 17 (Gln91; mutation) | 5′-NH2-(T)16-CTATCAGGCGATGGTG-3′ | 50.6 |
a
Probe lengths were limited to between 13 and 21 nucleotides. The probes specific for a given mutation were designed so that the base substitution was in the middle of the probe sequences. The predicted melting temperatures (Tm) of the probes were limited to between 48 and 52°C. All probes contained amino linkers at their 5′ ends for covalent attachment to aldehyde-coated slides. A spacer arm with a poly(T) 16-mer was inserted between the specific probe sequence and amino linker molecule at the 5′ end to decrease steric interference on the biochips.