PCR Amplification and Sequencing of C. jejuni porA fragment
PCR Amplification
A pair of oligonucleotide primers (MOMP-1 and MOMP-2) have been designed using an alignment of 24 C. jejuni and C. coli porA sequences available from GenBank. The primers are located within highly conserved regions and are designed to amplify a DNA fragment approximately 630 bp long. The precise amplicon size generated will vary with the genotype of the target isolate.
MOMP-1 5'-GAT GGT TTA ACT CTA GCT GC-3' MOMP-2 5'-TGA GAA GTT AAG TTT TGG AGA G-3'
These conditions have been optimised using Qiagen HotstarTaq DNA polymerase and Applied Biosystem 9600 or DNA engine thermocyclers; 96 well plates or 0.2ml tubes. 25 µl reactions contain each of the primers at a final concentration of 0.02 µM and dNTPs at 0.02 mM.
Cycling parameters:
| 1 cycle: | 95 °C for 15 min |
| 35 cycles: | 94 ºC for 30 sec |
| 50 °C for 30 sec | |
| 72 ºC for 1 min 30 sec | |
| 1 cycle: | 72 °C for 5 min |
| Hold: | 4 °C |
After PCR, the amplicons are precipitated using 60 µl PEG 8000 / 2.5M NaCl, washed twice using 70 % EtOH, dried and resuspended in 15 µl molecular biology grade water.
Sequencing
We sequence the amplicons in both directions using primers MOMP-1 and MOMP-2.
Each 10 µl sequencing reaction contains:
2 µl template DNA
4 µl 0.67 µM MOMP-1 or MOMP-2 (final concentration of 0.27 µM)
0.25 µl Big Dye (Applied Biosystems)
1.875 µl 5x reaction buffer (Applied Biosystems)
1.875 µl molecular biology grade water (Sigma)
Cycling parameters:
| 30 cycles: | 96 ºC for 10 sec |
| 50 °C for 5 sec | |
| 60 ºC for 2 min | |
| Hold: | 4 °C |
The extension products are precipitated using 50 µl EtOH and 2µl NaOAc pH 5.2 (7ml EtOH plus 280 µl 3M NaOAc pH 5.2 is sufficient for a 96 well plate), washed once with 150 µl 70% EtOH, dried and analysed using a 3730 Applied Biosystems Sequencer.
(Further details of the procedures for precipitation and washing can be found at http://pubmlst.org/neisseria/mlst-info/ nmeningitidis/nmeningitidis-info.shtml).