PCR-based Single-base Mutation Detection Assisted By T4 DNA Ligase

Single nucleotide polymorphisms(SNPs)are the most common human heritable variations,including single base transition,inversion,etc.,accounting for about 80%of all kinds of polymorphisms.The number of SNP sites in the human genome may reach 3 million,and it is closely related to all kinds of human diseases.Therefore,the accurate detection of SNP becomes particularly critical,and has gradually become a hot spot in the research of chemical biology,medicine and other frontier disciplines.In order to realize the detection of single-base mutation,sequencing detection and non sequencing detection have developed rapidly in recent years.The cost of next-generation sequencing has been greatly reduced with the rapid development of sequencing technology,but some drawbacks of sequencing technology,such as large nucleic acid concentration requirements,long analysis time,and the accuracy of detection is limited due to the depth of sequencing and the selection of nuclease,which makes the detection of low abundance mutation more difficult.With the concept of early diagnosis proposed,the next-generation sequencing technology can not meet the requirements of detection.Therefore,the development of simple,rapid and accurate non sequencing detection method of low abundance single base mutation has gradually become a new hot direction.The non sequencing detection methods of SNPs are mainly divided into two points: the first is the need to identify SNP sites,and the other is amplification to realize the detection of low abundance mutations.DNA ligase plays a very important role in the metabolism of life.It can catalyze the formation of phosphate diester bond between 3 '-terminal hydroxyl group and 5'-terminal phosphate group of DNA sequence,which is the last step of DNA replication,repair and recombination.Therefore,we selected DNA ligase to identify SNP sites,amplified by PCR technology,real-time fluorescence quantitative,to achieve accurate detection of SNP sites.T4 DNA ligase derived from T4 bacteriophage is a tool enzyme directly used in many experiments due to its wide application range,mild reaction conditions,high efficiency and low cost.Considering all aspects,we decided to use T4 DNA ligase-assisted q-PCR method for SNP low abundance detection.We use the single-base mutation recognition function of T4 DNA ligase,optimize its experimental conditions,which ultimately leads to different yields of the secondary templates N1-N2 required for PCR preparation in the ligation reaction stage.The two samples are used for subsequent PCR experiments.If there is a difference in the amplification curve,the samples with mismatched base pairs can be visually identified,and single-base mutation detection can be realized.We call this low-abundance single-base mutation detection technology of T4 DNA ligase-assisted PCR as L-PCR.The detection of dozens of mutant genes related to lung squamous cell carcinoma confirmed the feasibility of our L-PCR method to detect low abundance of mutant genes,and at least 0.1% of mutant genes can be detected.