Mutation Sensitive Switch Assays For Preeclampsia-associated AGT Polymorphism

Objective:This study was to investigate the mutations of the AGT gene(T174M and M235T)by using a mutation sensitive molecuLe "On/Off" system composed of a suLfonylated base-specific primer-coupled high-fidelity enzyme.At the same time,real-time quantitative PCR technique was used to determine the presence or absence of two hotspot mutations accurately and rapidly by amplification curve and melting curve.The mutation was also semi-quantitatively analyzed.Methods:The PCR product fragment containing the two codons was cloned into pMD19-T vector and transformed into E.coli TOP10 competent cells.The vector universal primers and PCR primers were used to identify the bacterial supernatants.The correct clones were screened out and confirmed by sequencing analysis.The wild-type plasmids of AGT gene were obtained.The PCR products were cloned and sequenced.The mutant template were cloned and sequenced.Then the two mutated sites were set as the detection targets,respectively,the 3’end of the wild-type gene or mutant gene locus paired suLfide-modified primer,the use of high-fidelity DNA polymerase mutation-sensitive molecuLe switch,The wild-type plasmids of AGT gene and two kinds of mutant plasmids were detected and analyzed by gel imaging system.Tissue or blood samples were collected from normal subjects and patients,clinical samples were evaluated.Samples were screened for two mutations of the AGT gene to evaluate the sensitivity of high fidelity enzyme-mediated sensitivity of molecuLar switches.Results:Two mutations in the AGT gene were screened for tumor samples.In the high-fidelity DNA polymerase-mediated PCR system,only complete primer pairs were extended,and incomplete primer pairs were not extended,i.e.,the PCR products of the mutant were only amplified from the paired mutant template,and no amplification products were detected in the wild-type template.Although the assay depends on the device,considering the current real-time PCR equipment has been widely equipped in the hospital,genetic analysis can provide resuLts within two hours,which may be helpfuL in cases of pre-eclampsia and other conditions in the emergency room or in the intensive care unit.Conclusions:In the present study,a DNA polymerase-mediated mutant-sensitive switch was used in two important AGT polymorphism analys.The resuLts showed that the mutation-sensitive switch was sensitive and specific for the detection of clinically relevant genotypes.These new assays are easy to use and cost effective and can be combined with other platforms such as real-time PCR to detect mutations test method to show AGT gene mutations(T174M and M235T).