We are identifying several unknown bacteria from water samples. The original protocol we are using to identify the unknown bacteria was based on a nested PCR by Sauer et at., 2005 using universal primers. When we tried the nested PCR our results were not good. But when we used the inner segment PCR primers to do a direct PCR and sequence the products we seemed to get good results but there are 2 main problems:
1. We are not sure it is right to use inner primers meant for a nested PCR to do a conventional PCR
2. The product size by the methodology was supposed to be 287 bp but ours was about 300-310 bp for all the samples we did.
Please, can someone clarify whether we are on track or not and how do we explain our findings. Many thanks
Hi pwaveno,
I do not think that using the internal primers isan issue. They were probably first used when not much genomic sequence was available to clean up a badly amplified product but as more sequence is posted you can be more confident as to how good they are and it is good evidence that they amplify clean sequences that sequence well. if a primer set amplifies well and sequences the right thing it is a good primer set
hi,
the answer is that often you can use nested primers as primary primers but sometimes you cannot. Nested primers can often be very poor primers with low annealing temperatures and great homology with other sequences in the genome because the first round of pcr amplification has hugely enriched the correct sequence so that even poorly designed nested primers will mainly anneal to the correct sequence and amplify only what you want. If they are used as the primery oligo set, however, all sites on the target genome will have an equal chance of being amplified so the amplification will be messy. It sounds like yours are annealing better to the wrong site and amplifying the wrong thing which is checkable either by sequencing (best) or by restriction digest since you know the correct sequence and where it cuts with all enzymes. If you Blast the primers you will prbably identify where else the internal primers will amplify..If you are unlucky one of the nested primers may have homolgy with a repeat sequence in the genome and could amplify on its own in forward and reverse direction and only works as a nested primer because the other primer adds the specificity to the amplification
Many thanks Prof. Paul Rutland for your response. After using the primers in direct PCR and getting good bands, the product was purified and sequenced using Sanger sequencing. The sequences were blasted in NCBI blast and Sepsi blast and most seem to have high similarity to other bacterial sequences with identity scores of 98-100 percent for most sequences which was what we wanted. Because we set out to identify unknown bacteria. But am worried that our methodology may be questionable because of the use of nested PCR primers in a direct PCR.
Dr. Singh thanks for your contribution. The nested PCR protocol primers were also universal primers but used in a nested PCR but we used the inner in a direct PCR. Thanks
Hi pwaveno,
I do not think that using the internal primers isan issue. They were probably first used when not much genomic sequence was available to clean up a badly amplified product but as more sequence is posted you can be more confident as to how good they are and it is good evidence that they amplify clean sequences that sequence well. if a primer set amplifies well and sequences the right thing it is a good primer set
a late thought Pwaveno,
if you get slightly too large but correct sequence ( by Blast) with nested omly and also with outer primers then nested primers then it is likely that the Sauer paper is wrong either because the sequence has changed since 2005 or maybe just a typing error. I think if blast gives you a larger size now then your larger size is correct unless with cheaper primers you have made the primers longer then you are probably both in agreement
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