Hi
I would like to know more about the reason for having phosphorothioate bond in both the 3' end of the adapter and PCR primer.
In the case of the adapter: Is it correct that this can prevent nuclease degredation of the "T" overhang?
But why the PCR primer needs this? I can only guess that the DNA polymerase Phusion can not remove the thio "T" from the pcr primer. So if the pcr primer anneal to a self-ligated adaptor, which might not be resulted from a T-A type ligation, Phusion will detected it as a mismatch. Since it is proof-reading, it will try to remove the mismatching "T" from the PCR primer. If it is not able to cew back the thio "T"... then everything make sense.
Cheers
sz
I would like to know more about the reason for having phosphorothioate bond in both the 3' end of the adapter and PCR primer.
In the case of the adapter: Is it correct that this can prevent nuclease degredation of the "T" overhang?
But why the PCR primer needs this? I can only guess that the DNA polymerase Phusion can not remove the thio "T" from the pcr primer. So if the pcr primer anneal to a self-ligated adaptor, which might not be resulted from a T-A type ligation, Phusion will detected it as a mismatch. Since it is proof-reading, it will try to remove the mismatching "T" from the PCR primer. If it is not able to cew back the thio "T"... then everything make sense.
Cheers
sz