Taqman Probe Couple With DNA-binding Dye Based COLD-PCR Enables Rapid Screening Of Low Abundance KRAS Mutation In Blood

Early detection of genetic mutations is increasingly recognized to play an important in clinical decision for individualized therapy.Lung cancer,among many other cancers,has often been found to harbor KRAS mutations,which is of relevance to clinical treatment.Currently,Sanger sequencing after PCR amplification is the method of choice to detect mutations,but this method can only detect mutations when the mutated sample is higher than 15-20%;furthermore,the procedure is cumbersome and expensive.Other methods also have various shortcomings.Then it would be of great help to have a method that is simple,easy and fast operation,low cost,good accuracy for the preliminary screening of mutations.Here we present a COLD-PCR based qPCR method that combined wild-type Taqman probe and DNA binding dye for mutation detection: COLD-PCR can selectively enrich mutations in a high background of wild-type sequence by changing the denaturation temperature in PCR;both wild-type probe and DNA binding dye were added in the qPCR reaction system.In principle,the method described above can enrich and filter all types of mutations that do not match wild-type probe simultaneously,either known or unknown,single or multiple,point mutation or a long string of mutations.This method would be helpful to the preliminary screening of mutations.The feasibility of this technique is demonstrated by detecting KRAS mutations in blood from lung cancer patients.This study aimed to establish a COLD-PCR based qPCR method that combined wild-type Taqman probe and DNA binding dye,then verify its feasibility,and finally apply this method for preliminary screening of KRAS mutations in clinical samples.Methods:1.Cell lines containing wild-type and mutant-type KRAS were chosen,followed by design and synthesis of primers and probes specific to wild-and mutant-type KRAS gene(all marked by ROX).After extraction of DNA from the respective cell lines,regular PCR and Sanger sequencing was performed to verify the genotype.2.DNA-binding dye based qPCR was applied to amplify DNA from wild-type and mutant-type KRAS respectively,with the denaturation temperature gradually reduced.Critical denaturation temperature(Tc)for COLD-PCR was identified when mutant-type KRAS underwent amplification while wild-type KRAS did not.3.Normal qPCR using both DNA-binding dye and wild-type Taqman probe as detection method(Method 1)was established and optimized by adding Taqman probe specific to wild-type KRAS was added to DNA-binding dye based qPCR reaction system.4.On the basis of method one,cycling condition in normal PCR was changed to that in COLD-PCR,trying to establish COLD-PCR using wild-type probe +DNA-binding dye as detection method(Method 2).5.Both Method 1 and Method 2 were applied to amplify and detect standard sample containing different proportion of mutant KRAS.The fluorescence from EVAgreen dye and ROX marked probe was recorded and their ratio EVA/ROX was calculated,followed by analysis of mean value and standard deviation means±SD,(n=3).The limit of detection was defined as the minimum proportion of KRAS mutation for which EVA/ROX could be reliably detected and differentiated from the EVA/ROX of pure wild KRAS gene.6.Replace the wild-type KRAS specific probe with mutant-type KRAS specific probe in both Method 1 and Method 2 and apply the method to detect standard samples containing different proportions of mutant KRAS gene.The fluorescence signal from Objective:DNA-binding dye and Taqman probe were recorded and their ratio calculated to verify the feasibility and reliability of the above two methods.7.Samples of peripheral blood were collected from 25 lung cancer patients;after extraction of DNA,screening of KRAS mutations was performed using the two methods described above.In the case when KRAS mutations were detected by either Method 1 or Method 2,the residual DNA was amplified with normal PCR kit using regular PCR or COLD-PCR,followed by Sanger sequencing for validation.Results:1 The sequencing results verified that the two cell lines contained their expected gene type.Tc in COLD-PCR was 80.5℃.2 When applied to detect standard samples containing different proportions of mutant KRAS,according to the value of means±SD for EVA/ROX,the limit of detection was 10% for Method 1 and 5% for Method 2,respectively,which means COLD-PCR enriches mutations better than regular PCR.3 With wild-type probe replaced by mutant-type specific probe in both Method 1and Method 2,the fluorescence signal and ratio of the DNA-binding dye to mutant-type probe was observed to be in agreement with expectations,demonstrating the feasibility of the above two methods.4 When applied to the detection of 25 blood samples,Method 1 failed to detect KRAS mutation,while Method 2 successfully detected one case of KRAS mutation.According to the value of EVA/ROX of the sample,and comparing it with the EVA/ROX value of the standard sample using Method 2,the proportion of mutant KRAS in this sample could be half quantitatively estimated to be between 5% and 10%.5 The blood sample,from which mutant KRAS had been detected using Method2,was found to be positive in mutation in KRAS after COLD-PCR and sequencing,while negative after normal PCR and sequencing.This result not only verified the reliability of Method 2,but also showed that COLD-PCR could enrich mutations better than normal PCR,which can improve the sensitivity of downstream sequencing procedure to detect mutations.Conclusion:1.We have successfully established a COLD-PCR based method to detect genetic mutations using wild-type Taqman probe in combination with DNA-binding dye in one reaction system.It has been demonstrated to detect as low as 5% of mutant KRAS in a background of wild-type KRAS,which favorably compared with 15-20% of normal PCR after Sanger sequencing.This method is simple and easy to operate,cheap,reliable and does not require complex instrument.It can be applied to liquid biopsy such as blood,and is non-invasive.In principle,it can detect multiple mutations simultaneously,and may find applications in preliminary screening of mutations.2.Compared with normal PCR,COLD-PCR can enrich mutations more effectively,improving the sensitivity of downstream sequencing procedure to detect mutations.After initial detection of KRAS mutations by Method 2,COLD-PCR in combination with Sanger sequencing can be applied to achieve accurate identification of mutations.