For 16S rRNA sequencing to identify bacterial isolates there are several universal primers (forward and reverse) such as 8F, 1492R, 27F, 928F, 336R etc. Are these primers code for some gene? How can we detect or identify this?
Thanks.
I ‘am not quite sure whether I got your question correctly. The primers do not code for any gene. Rather, the number within the primer name corresponds to the position on the E. coli 16S rRNA gene. 8F for instance refers to position 8 (forward direction), while 1492R refers to position 1492 (reverse direction). Depending on what you are interested in to amplify (i.e. which amplicon size for sequencing) you may choose 8F and 1492R (than your PCR would cover almost the entire gene) or – sufficient in many cases – 27F and 907R. With the latter combination you’ll get more than half of the gene amplified. Since numbering corresponds to the E. coli reference, you may be prepared to get amplicons which are not exactly the expected size with the bugs you are working with. The length of the 16S rRNA genes can vary significantly, depending on the species/genus.
I ‘am not quite sure whether I got your question correctly. The primers do not code for any gene. Rather, the number within the primer name corresponds to the position on the E. coli 16S rRNA gene. 8F for instance refers to position 8 (forward direction), while 1492R refers to position 1492 (reverse direction). Depending on what you are interested in to amplify (i.e. which amplicon size for sequencing) you may choose 8F and 1492R (than your PCR would cover almost the entire gene) or – sufficient in many cases – 27F and 907R. With the latter combination you’ll get more than half of the gene amplified. Since numbering corresponds to the E. coli reference, you may be prepared to get amplicons which are not exactly the expected size with the bugs you are working with. The length of the 16S rRNA genes can vary significantly, depending on the species/genus.
These primers are suitable for environmental bacteria:
16F27 (5′-CCAGAGTTTGATCMTGGCTCAG-3′) and 16R1488 (5'-AGAGTTTGATCMTGGCTCAG-3')
There are positions well conserved. Of course you have to play with PCR conditions to avoid inespecificity. We have amplified 16S genes from most of the isolates obtained.
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