Amplification and sequencing of double AB infected individuals

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.