| Propionibacterium
acnes - coming soon
The nine provisional loci and the primers and conditions
used for PCR;
The provisional P.acnes MLST scheme uses internal fragments
from 9 housekeeping genes spread around the genome;
lac (L-lactate dehydrogenase)
cob (Cobalamin biosynthesis CobD/CbiB protein)
oxc (Cytochrome c oxidase subunit II)
coa (O-succinylbenzoate-CoA synthase)
zno (Zn-dependant alcohol dehydrogenase)
gms (Glutamyl-tRNA synthetase)
pak (Pantothenate kinase)
fba (Fructose bisphosphate aldolase)
cel (Transcription regulator CelR)
PCR Conditions:
There are 3 different annealing temperatures to cover the amplification
conditions of the 9 provisional PCR targets. As the final scheme
is refined and finalised 1 annealing temperature may cover
all targets. PCR amplification is carried out on chromosomal
DNA using an extension time of 30 seconds, and an annealing
temperature of 50C1, 52C2 or 55C3, with Qiagen Taq polymerase.
This is repeated for 34 cycles. As the same primers are used
for amplification and sequencing, it is important that only
a single DNA fragment is amplified in the initial PCR. This
may involve some optimisation of the annealing temperature.
1 – cob (primers at 50pmol/mL concentration).
2 – Annealing temperature of lac, oxc, fba.
3 – Annealing temperature of coa, zno, gls, pak, and
cel.
The primer sequences for each PCR are;
cob up - 5’ CGA CGC CGC CGA GAT AG 3’
cob dn - 5’ TCA GGC CGC CGT AGA ACA 3’
lac up - 5’ GCC GCA GCC TTG GGA CTC T 3’
lac dn - 5’ GAA ATG CTG TCG CCC CGT G 3’
oxc up - 5’ GTG CTG CCG GAA AAG TCG 3’
oxc dn - 5’ CAC CGG CGT CAG GAT TGT 3’
fba up - 5’ AGG ACC CGC TAT TTC AAC TCT CA 3’
fba dn - 5’ ACG CGG GTC GTA CAT CTT CTT 3’
coa up - 5’ GCG GGA ATC GAG GGT GCT A 3’
coa dn - 5’ AGG GCC GCC GCT AGA TAA GTA 3’
zno up - 5’ CGC CGG CAT CAC CAC CTA TT 3’
zno dn - 5’ TCT CAC ATC GCC CGC AAC C 3’
gls up - 5’ CCG CCT CAC CGT CCA GCA 3’
gls dn - 5’ CAC ATC GAG AAC CGC ATC ACTC 3’
pak up - 5’ CGACGC CTC CAA TAA CTT CC 3’
pak dn - 5’ GTC GGC CTC CTC AGC ATC 3’
cel up - 5’ GCC GAC GTT TTC TAC AGT GAG C 3’
cel dn - 5’ GGC GGT GAG GGT CCA TTC A 3’
For further information please contact Simon
O'Hanlon
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