Study On PCR Platform Mediated By Mesorhizobium Japonicum Argonaute Protein

The polymerase chain reaction（PCR）has significantly advanced modern biology development.PCR has been widely used to test for COVID-19 in the past.Although many new techniques of nucleic-acid detection have emerged,quantitative PCR is still considered the gold standard for COVID-19 testing,as recommended by the World Health Organization.The combination of primer and template is an important step in PCR.The low efficiency of the combination of primer and template directly leads to the low sensitivity of PCR detection platform.Prokaryotic Argonaute proteins are characterized by highly efficient guide DNA/RNA-directed binding to target nucleic acids with high fidelity.Employing this feature,we designed an argonaute-facilitated PCR platform to solve the problem of low sensitivity of PCR technology.We selected an argonaute derived from Mesorhizobium japonicum（MejAgo）which had three characteristics:（1）exposure the 3’end of guide DNA when binding of DNA guide,（2）targeted binding ability,（3）does not carry nuclease activity.MejAgo allows two-step PCR to use extensively,each reaction cycle of the MejAgo-PCR platform consists of a denaturing step and a polymerase-mediated extension step,omitting the annealing step required by the traditional PCR.It is proved that MejAgo can promote the binding efficiency of primers and templates,thus enhancing the sensitivity of traditional PCR platform in nucleic acid detection.The specific contents of this study are as follows:（1）The structure analysis and in vitro cleavage activity verification of MejAgo protein proved that was suitable for Ago-PCR,and the optimal induction temperature of MejAgo was 18℃,ensuring the stable output of high-quality protein that could be used for MejAgo PCR;（2）It was found that the final concentration of MejAgo required in the MejAgo-PCR amplification system was450-1300nm;Using 5’phosphorylated guide DNA is more stable;The length of primers ranged from 27nt to 50nt,and the length of the target product selected for detection was less than 450bp;It can detect double stranded linear DNA,single stranded circular DNA and double stranded circular DNA substrates;BSA with a final concentration of100ug/m L was more stable in the amplification system;Incubating MejAgo with primers in advance and then performing two-step variable temperature amplification will improve the efficiency of MejAgo in promoting primer template pairing;MejAgo-PCR two-step amplification with variable temperature for 50 cycles can detect 6copies/20u L of the target sequence in 1h10min,and mixing 300ng of 293T cell genome into the system will not affect the detection of low copies of the target sequence;MejAgo-PCR needs to rely on metal Mg²⁺concentration of 2-6m M;MejAgo can complete the amplification reaction with Taq DNA polymerase,Phanta Super-Fidelity DNA polymerase,One Taq DNA polymerase,Long Amp Hot Start Taq DNA polymerase and Deep vent DNA polymerase respectively in the commercial buffer provided by different polymerases.