Great article! From what you have read, how might error rates and fidelity of pipolB compare to conventional high-fidelity polymerases, and what implications would that have for downstream sequencing?
Standard DNA polymerases tend to have built in proofreading activity, but pipolins don't seem to have an equivalent feature. This is definitely a concern, but likely something that can be fixed with techniques like directed evolution to arrive at a less error-prone polymerase. For all we know, there could be a tradeoff between function and fidelity, but we don't know enough about pipolins yet.
According to the patent that the Pasteur Institute filed for this discovery (which includes the use case for primerless PCR), there is a specific peptide sequence which is responsible for its primerless replication. It's probably possible to just insert the motif into a high fidelity polymerase like Pfu. Here's the patent: https://patents.google.com/patent/US20200263151A1/en
You also mentioned RNA and the necessity of RT, are we sure that all RNA in the sample is completely transcribed into DNA? Random primers work great for standard samples but how does it look for META samples where there should be more RNA in addition to a lot of DNA?
And the second question, can metagenomic samples be prepared in a way that would remove the most represented (and therefore probably already identified) DNA molecules, i.e. to leave only what standard methods have a problem with in the sample
Great article! From what you have read, how might error rates and fidelity of pipolB compare to conventional high-fidelity polymerases, and what implications would that have for downstream sequencing?
Thanks Manav, and great question!
Standard DNA polymerases tend to have built in proofreading activity, but pipolins don't seem to have an equivalent feature. This is definitely a concern, but likely something that can be fixed with techniques like directed evolution to arrive at a less error-prone polymerase. For all we know, there could be a tradeoff between function and fidelity, but we don't know enough about pipolins yet.
According to the patent that the Pasteur Institute filed for this discovery (which includes the use case for primerless PCR), there is a specific peptide sequence which is responsible for its primerless replication. It's probably possible to just insert the motif into a high fidelity polymerase like Pfu. Here's the patent: https://patents.google.com/patent/US20200263151A1/en
Great article, thx for that.
You also mentioned RNA and the necessity of RT, are we sure that all RNA in the sample is completely transcribed into DNA? Random primers work great for standard samples but how does it look for META samples where there should be more RNA in addition to a lot of DNA?
And the second question, can metagenomic samples be prepared in a way that would remove the most represented (and therefore probably already identified) DNA molecules, i.e. to leave only what standard methods have a problem with in the sample