A New Method For Digital Detection Of Genetic Markers

With the rapid development of biomedicine,the significance of genetic markers in modern medicine is becoming more and more important.Many genetic markers have been found to be closely related to the occurrence and development of diseases,including genetic polymorphism,somatic mutation,copy number variation and abnormal gene methylation.By detecting these genetic markers,it becomes possible to predict the risk of disease,early diagnosis of disease,monitor the efficacy of treatment or assess the prognosis.However,because the genetic markers are often very small changes in DNA sequence,may be only single base change or a fraction of cell variation,which put forward a high demand on the detection of genetic markers.Digital PCR is a new generation of nucleic acid quantitative detection method based on single molecule PCR.The fundamental is that the target molecules are dispersed into a large number of micro reaction units,so that each reaction unit contains no more than one target molecule,in which the single molecule PCR amplification reaction was carried out independently.By detecting each reaction unit after the reaction,the absolute quantification of the initial nucleic acid target can be achieved.Digital PCR is superior to traditional real-time fluorescence quantitative PCR in quantitative accuracy and sensitivity,and plays an increasingly important role in nucleic acid detection.First,we use the digital PCR coupled with hydrogel beads-array to quantitative determine the methylation level of colorectal cancer related VIM gene from stool samples.This method is highly sensitive and specific and is expected to apply on the non-invasive screening of colorectal cancer.However,PCR amplification is a template-amplification based method in which a large number of amplification products consistent with the template molecule in sequence were generated,which can serve as templates for the next assay reaction.Thus,the contamination of amplified products will lead to the false-positive results.Differ from PCR and other template amplification methods,nucleic acid signal amplification is a method of triggering signal molecule amplification by template molecule.The number of template molecules themselves does not increase during the reaction,but the target specific signal molecules are cyclically amplified instead,making it an ideal way to avoid amplicons contamination.Based on the previous works,this study put forward a new idea of single molecule analysis combined with signal amplification and digital detection.First,the structure-specific invasive reaction is used to convert the target to the signal molecules which are independent of the template sequence.Then the signal molecules are further amplified by the cascade reaction,and produce the fluorescent signal,which is independent of the target sequence but specific to the target molecule.In order to achieve single molecule digital detection based on invasive reaction,we made some improvement against current problems of invasive reaction.The first problem is how to improve the specificity of invasive reaction for recognizing the single-base variation.While in theory,the single base difference at the invasive site can be identified,but in practice,we found that the non-specific reaction did occur on some cases,making it failing to detect the single base difference.In this regard,we proposed to introduce additional mismatched bases in the downstream probe of the invasive reaction.By optimizing the introduction position and the type of the mismatched base,we achieved a significant reduction in non-specific signal without affecting the specific reaction,and improved the specificity of the invasive reaction in single base differential detection.The second problem is how to reduce the background signal of "X" type structure in invasive reaction.In the conventional cascade invasive reaction,a non-specific structure called "X" type structure formed by the downstream probe and the fluorescent hairpin probe can also be identified and cleaved by FEN1 endonuclease,resulting in background signal.In the case of detecting low copy target,from which the positive signal produced is weak,the background signal will cause serious influence on the detection result,so that the positive and the negative result cannot be effectively distinguished.In order to overcome the background signal problem,we proposed a new type of cascade invasive reaction mediated by a controllable extension reaction.In this method,by introducing a spacer sequence between the hybridization sequence between the downstream probe and the fluorescent probe,the formation of the "X" type structure was avoided,and the background signal was significantly reduced.The two-step invasive reaction was bridged by a controlled extension reaction to form signal amplification.As the background signal was suppressed,the positive and the background signal could be distinguished;the detection sensitivity of the reaction was greatly improved.Base on this method,it is expected to further establish a single molecule level invasive reaction method.Finally,we applied the low background cascade invasive reaction based on the controllable extension reaction to the beads-emulsion system,and significantly reduced the background signal in the negative reaction unit.Using the established low background cascade nucleic acid invasion reaction based on the beads-emulsion system,we have achieved detection of the single target molecule,and the positive signal is significantly different from the background signal of the negative reaction unit.The digital signal amplification method we have established will provide a new choice for digital nucleic acid analysis.