I recently designed a pair of primers capable of amplifying 12 Chlamydia species with a target fragment length of 184bp, which was confirmed to be Chlamydia-specific by NCBI primer BLAST. I used this primer pair to test several DNA extracted from chicken cloacal swabs, and several positive DNA amplification products I did Sanger sequencing, is the sequencing results and Chlamydia only 84-87% homologous sequence, and some other bacteria homologous sequence up to 98%, but this primer pair can not match to these high homologous bacteria, forward and reverse primers have at least two base mismatches. What causes this phenomenon and is there any way to verify the specificity of this primer pair?
Primers can still amplify a target, even with a few mismatches. In fact, a mismatch at the 3' end is sometimes tolerated.
The only way to show specificity is to show that your primers DO NOT amplify other targets.
You've demonstrated that your primers are in fact not specific. A better strategy would be to find a region that is unique to Chlamydia and not found in other microbes.
Good luck!
I agree with Katie A S Burnette that the best method is amplifying a chlamidia specific region and primers with mismatches are commonly used in pCR. If you are using mismatched primers then it is necessary to use the most stringent annealing conditions to avoid unwanted amplification. Use the highest temperature that works (run a gradient pcr if possible) even though it give a low yield. You could also try running a DMSO gradient up to 10% dmso which will stop some mismatched primers from binding and help the correct primer/template match to become the most common amplimer
If you mean that you cannot see the primer sequence when viewing the .abi sequence file then you never get any sequence under the primer or in the next 30 bases from the primer at one end and at the other end the sequence will be present but in the reverse complimentary orientation
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