Amino-modified Deoxynucleotides For Long-range PCR Research

Since its inception,DNA polymerase chain reaction(PCR)has quickly attracted attention due to its simple operation and strong sensitivity.It plays an important role in genetic engineering,genetic diagnosis and disease detection.One of the development directions of PCR technology is the amplification of long fragments of DNA.Long-range PCR is characterized by the length of its amplified fragments,but current amplification lengths cannot completely amplify genes in organisms.The long-range PCR reaction lasts longer,the thermal stability of the reaction substrate is more demanding.At present,several commercial DNA polymerases have met the thermal stability requirements,but the thermal stability of dNTPs in the reaction has been ignored.Therefore,the development of new thermally stable dNTP analogs has important research significance.Studies have found that the two hydroxyl groups on the terminal phosphate of dNTP are the main reasons for its instability.Considering that the terminal phosphate group is far away from the catalytic center of the DNA polymerase and has little effect on the enzyme reaction activity,this paper designs and synthesizes multiple dNTP terminal phosphate modifications in order to enhance its thermal stability,it can be recognized and utilized by DNA polymerase for long-segment PCR amplification.The main research contents of this paper are as follows:1)Considering the thermal stability of the compound and its effect on the reaction rate of DNA polymerase,this article replaces a hydroxyl group on the terminal phosphate of dNTP with an amino group to synthesize phosphoramide compounds.We tried a variety of synthetic methods and successfully synthesized four target compounds,dATP-NH2,dTTP-NH2,dCTP-NH2,and dGTP-NH2,and found a synthetic route with relatively high yield,with a yield of 40%-60%.2)We verified its thermal stability by simulating the PCR environment.After 48 hours of constant temperature incubation at 75?,dATP-NH2 remained 69%,while dATP only remained 15%,indicating that its thermal stability was greatly improved compared with natural dNTP.While studying the thermal stability and analyzing the hydrolysates of the two,it was found that the hydrolyzed compounds were both dADP and dAMP,and the proportion of dADP was higher than that of dAMP during the entire experiment,indicating that the dNTP hydrolysis starts from the terminal phosphate and drops off the phosphate groups one by one.3)Using dNTP-NH2 as the raw material,using different DNA polymerases for amplification,all the target amplification products are obtained.Prove that the synthesized compound can be recognized and utilized by a variety of DNA polymerases;use the synthesized compound to explore the effect of PCR extension time on the amplification of the target gene.It is confirmed that as the extension time increases,the amplification of the target gene increases,indicating that dNTP-NH2 is used as Substrates can compensate for the slightly lower efficiency of the enzyme reaction by increasing the extension time.4)Using dNTP-NH2 as the substrate for PCR reaction,the 1 kb,3 kb and 6 kb and 10 kb fragments were successfully amplified,indicating that dNTP-NH2 can be used not only for conventional PCR,but also for long fragment PCR reactions.Different DNA polymerases were used to successfully amplify the target gene in long-range amplification(6 kb),indicating that dNTP-NH2 has a broad application prospect in long-range amplification.In summary,the dNTP-NH2 synthesized in this paper has significantly improved thermal stability,can be recognized by a variety of DNA polymerases and performs well in PCR amplification,indicating that dNTP-NH2 has broad application prospects in long fragment amplification.