Simultaneous Detection Of Three Genotypes Of E545K Mutations For PIK3CA Gene Using TaqMan Probe In Combination With Dye Based Probe In Combination With Dye Based Droplet Digital PCR

Objective The current method to detect single mucleotide polymorphisms(SNP)using dd PCR is mainly based on duplex dd PCR,which exploits a pair of probes specific for wild and mutant genotypes.However,this method can only detect a genotype that is already known.In order to break this limit and improve the use fulness of dd PCR,a dd PCR-based method is established that utilizes wild-type probe in combination with DNA binding dye,and three genotypes of E545 K mutations for PIK3 CA gene are detected using this method.Methods This is methodological study.First,the method was established;then comparison with other methods was performed for statistical analysis to demonstrate the reliability of the new method.1)Cell lines containing the wild-and mutant-type E545 K for PIK3 CA gene were chosen;then primers and a pair of probes specific for wild-and mutant-type PIK3 CA gene were designed and synthesized.2)After PCR amplification of the nucleic acids extracted from the cell lines,sequencing of PCR products were performed to determine the genotype of PIK3 CA in E545 K for each cell line.3)Real-time quantitative PCR(q PCR)was performed to check the specificity of the primers and probes;then a method is preliminarily established utilizing wild-type probein combination with DNA binding dye;optimization was followed to maximize the difference influorescence from the samples of three genotypes such that the method could be applied to distinguish the three genotypes.4)The q PCR reaction system established above was then applied to dd PCR;after optimizing the method using wildand mutant-type probes based duplex dd PCR and another method using wild-type probe in combination with DNA binding dye,detection of three genotypes of PIK3 CA was performed to make sure that each genotype can be distinguished clearly.5)Apply the two dd PCR methods described above to detect a mixture sample containing the three genotypes,and quantitatively obtain the copy number of each genotype;t test was applied for the copy number of each genotype,followed by analysis of P value to judge whether the data obtained by the two methods differ significantly,which verifies the reliability of our new method.Results1)After optimization of the q PCR reaction system and reaction conditions,the optimal annealing temperature is 62℃,with the system amplication efficiency(E)to be0.918.High amplification efficiency can reduce the difference in fluorescence intensity among various droplets caused by different amplification efficiency in large amount of droplets,which might result in inaccurate results.2)A test using double probes-based duplex q PCR was performed for each of the three genotypes,and analysis of the amplification curve showed that both the wild-type and mutant-type probe exhibited good specificity.3)A method based on q PCR combining wild-type probe and DNA binding dye was preliminarily established and then applied to detect pure samples from the three genotypes;samples with the same amount of DNA input was detected for optimization that maximized the difference of fluorescence intensity for wild type probe(VIC).4)The optimized double probes-based dd PCR could be used to detect the mixture sample containing three genotypes,and each of the three genotypes can be clearly distinguished with the corresponding copy number counted based on the distribution of the fluorescence intensity of droplets.5)The optimized dd PCR method using wild-type probe in combination with DNA binding dye could also simultaneouslydistinguish each of the three genotypes in a mixture sample with the respective copy number counted based on the distribution of fluorescence intensity of droplets.6)The copy number of each of the three genotypes in mixture sample was obtained using both double probes-based dd PCR and wild-type probes in combination with DNA binding dye based dd PCR;t test was performed respectively,with wild type group:P=0.954>0.05,heterozygous mutations type group: P=0.451>0.05,homozygous mutations type group: P=0.308>0.05.The data demonstrated that these two detection methods showed no statistically significant difference when applied for detection.Conculsion By combining wild-type probe and DNA binding dye,we successfully established a dd PCR method that can simultaneously detect the copy number of three genotypes of PIK3 CA E545K mutation in one PCR reaction.This method used only one wild-type probe,but can detect any SNPs or other types of mutations that did not match the sequence of the wild type probe.It broke the limits of the ordinary double-probe approach that can only detect the mutation for which the mutant-type probe was disigned.This method reduces the cost of mutation test and might find applications in mutation screening.