PRIMERS and PCR conditions for N. gonorrhoeae MAST

The PCR conditions that we use in our laboratory may have to be modified slightly in other laboratories due to differences in PCR machines etc. Optimisation of the annealing temperature in your laboratory using a range of temperatures around that suggested below is often useful (temperature-gradient PCR is a convenient way of doing this), to provide a temperature that maximises the amount of the amplified product and minimises spurious amplification of any other non-specific products. Optimisation is important as the same PCR primers are used for the initial amplification of the internal fragments of por and tbpB and for the sequencing reaction - therefore the initial amplification must result in the specific amplification of only the desired gene fragment.

Por forward 5’ ³⁵⁰CAA GAA GAC CTC GGC AA³⁶⁶ 3'
(based on the numbering from strain MS11 - the primer is between loops 1 and 2)

Por reverse 5’ ¹⁰⁸⁶CCG ACA ACC ACT TGG T¹⁰⁷¹ 3'
(based on the numbering from strain MS11 – the primer is between loops 7 and 8).

The GenBank accession number for the por sequence of strain MS11 is M21289.

TbpB forward 5’ ¹⁰⁹⁸CGT TGT CGG CAG CGC GAA AAC ¹¹¹⁸ 3’
(based on the numbering from strain UU1008)

TbpB reverse 5’ ¹⁶⁸⁶TTC ATC GGT GCG CTC GCC TTG¹⁶⁶⁶ 3’
(based on the numbering from strain UU1008).

The GenBank accession number for the tbpB sequence of strain UU1008 is2286066.

PCR conditions

PCR reactions in our laboratory are in 50µl volumes and are carried out in 96 well microtitre plates with Qiagen Taq Polymerase, using a PTC-200 DNA engine (MJ Research Inc) with an initial denaturation at 95oC for 4 minutes, followed by 25 cycles of 950C for 30 seconds, 580C for 30 seconds for por and 620C for tbpB, 720C for 1 minute, followed by 720C for 10 minutes and cooled to 40C and held. The amplified DNA fragments are precipitated using 20% PEG 8000, washed twice with 70% ethanol, dried and resuspended in sterile water. The sequencing reaction uses the same primers for each strand of the gene fragment as used for PCR amplification using BigDyeTM terminator (Applied Biosystems). The sequencing reaction product is purified by precipitation with sodium acetate and 95% ethanol and washed twice with 70% ethanol.

The sequencing is carried out on an ABI 3700 sequencer.

The forward and reverse sequences of each strain are aligned, edited and trimmed to a set length.

The por sequence is trimmed to 490 bp, starting from the following conserved region spanning from pre-loop 3 to loop 6:

TTGAA

The tbpB sequence is trimmed to 390 bp, starting from the conserved region:

CGTCTGAA

Click the name below to obtain a correctly trimmed sequence for that locus

Por | tbpB |