Use the following protocol for specific amplification and sequencing of A and B Wolbachia strains in host individuals carrying a standard A-B double infection.
Since individuals of the same species but coming from different populations can harbor different Wolbachia strains, please always use DNA extracted from a single individual, or from multiple individuals derived from a single female. For each strain perform PCRs using the same DNA for amplification of all five genes. This will assure that all five alleles corresponding to a single strain.
Primers
Gene | Supergroup specific | Primer name | Primer sequences (5'-3') | PCR Annealing T (°C) |
---|---|---|---|---|
gatB | B-specific | gatB_BspecF1 | TAAGAATCGCAAGAATTCAC | 62 |
gatB_R1 | TGG YAA YTC RGG YAA AGA TGA | |||
A-specific | gatB_AspecF1 | TTTAGAGCAAGATGCAGGRAAGAGCG | 64 | |
gatB_R1 | TGG YAA YTC RGG YAA AGA TGA | |||
coxA | B-specific | coxA_BspecF1 | ATACCCACCTYTRTCGCAAA | 54 |
coxA_R1 | CT AAA GAC TTT KAC RCC AGT | |||
A-specific | coxA_AspecF1 | ATACCCACCTTTATCACAGG | 56 | |
coxA_R1 | CT AAA GAC TTT KAC RCC AGT | |||
hcpA | B-specific | hcpA_F1 | GAA ATA RCA GTT GCT GCA AA | 55 |
hcpA_BspecR1 | TTCTTTGTCGCTMACTTYAATCAKG | |||
A-specific | hcpA_F1 | GAA ATA RCA GTT GCT GCA AA | 55 | |
hcpA_AspecR1 | TTCTARYTCTTCAACCAATGC | |||
ftsZ | B-specific | ftsZ_BspecF1 | AAAGATAGCCATATGCTCTTT | 59 |
ftsZ_BspecR1 | CATTGCTTTACCCATCTCA | |||
A-specific | ftsZ_AspecF1 | AAAGATAGTCATATGCTTTTC | 55 | |
ftsZ_AspecR1 | CATCGCTTTGCCCATCTCG | |||
fbpA | B-specific | fbpA_BspecF1 | GTTAACCCTGATGCTTACGAT | 58 |
fbpA_BspecR1 | CCRCCAGARAAAAYYACTATTC | |||
A-specific | fbpA_AspecF1 | TTAACCCTGATGCTTATGAC | 55 | |
fbpA_R1 | CCR CCA GAR AAA AYY ACT ATT C |
PCR protocol
This protocol is for guidance only and you may need to optimize it for your particular laboratory.
Perform PCR reactions in a 40 μL final volume using 2 μL DNA, H2O to volume and reagents at the following concentrations:
Reagents | PCR final concentration |
---|---|
Buffer | 1x |
dNTPs | 0.2 mM |
MgCl2 | 1.5 mM |
Primer F | 1 μM |
Primer R | 1 μM |
Taq DNA polymerase | 0.5 U |
Cycling conditions
- 94°C for 2 min
- 37 cycles at:
- 94°C for 30 s
- optimal annealing T for 45 s (as specified in the primer table above)
- 72°C for 1 min 30 s
- 1 cycle at:
- 72°C for 10 min
- 4°C hold
After cycling, check that amplification was successful by running 5 μl of each reaction on an agarose gel, with size standards.
Purify the remaining 35 μl and sequence using both forward and reverse primers. A double coverage of each sequence is required.
Use the allele templates provided as reference to verify the exact range of nucleotides for each gene sequence.