Short-DNA Specific Blocker PCR for Efficient and Simple Enrichment of Cell Free Fetal DNAs with Short Lengths

As an inn ate characteristic,DNA length has attracted more and more atte ntion.The length of some import ant biomarkers such as ctDNA(Circulating tumor DNA)and cffDNA(Cell free fetal DNA)is shorter than that of cell-free DNA.Researchers have utilized the differe nee in length to in crease the abunda nee of ctDNA or cffDNA in total cell-free DNA to overcome the difficulties in detecti on due to their low abundance.

Shared-probe system:An accurate,low-cost and general enzyme-assisted DNA probe system for detection of genetic mutation

Enzyme assisted DNA probes are powerful tools in molecular diagnostics for their simplicity,rapidity,and low detection limit.However,cost of probes,difficulty in optimization and disturbance of secondary structure hindered the wider application of enzyme assisted DNA probes.To solve the problems,we designed a new system named shared-probe system.By introducing two unlabeled single stranded DNA named Sh1 and Sh2 as the bridge between probe and the substrate,the same sequence of dually labeled probe with stable performance was shared for different mutations,thus sparing the expense and time cost on designing,synthesizing and optimizing corresponding probes.Besides,the hybridization between Sh1 and the substrate could overcome secondary structures,which guaranteed the detection of different substrates.The performance and generality of the design were tested by low abundance detection in synthetic single DNA samples and the limit of detection was 0.05%for PTENR130 Q,EGFR-L858 R and 0.02%for BRCA1-NM007294.3.In genomic DNA samples,the limit of detection of 0.1%can be achieved for EGFR-L858 R,demonstrating the potential of clinical application in our design.

Thermodynamics-guided two-way interlocking DNA cascade system for universal multiplexed mutation detection

Detection of point mutations in driver genes is of great significance for the early diagnosis,treatment,and prognostic evaluation of cancer.However,current detection methods do not offer versatility,specificity,and rapid performance simultaneously.Thus,multiple mutation detection processes are necessary,which results in long processing times and high costs.In this study,we developed a thermodynamics-guided two-way interlocking DNA cascade system for universal multiplexed mutation detection(TTI-CS).This strategy is based on the DNA probe,which changes the thermodynamic balance of the DNA cascade by the designed bubble structure,thereby achieving a good distinction between mutant and wild-type DNA.The designed method greatly shortens the detection time through two-way intrusion.In addition,this method only changes two inexpensive trigger and bridge sequences,which replace the specific and expensive nucleic acid probes used in analyses based on traditional DNA probe methods,thereby enabling multiple detections.We performed the detection of synthetic single-stranded DNA for the five mutation points and successfully detected in endometrial cancer specimens.The detection limit of this method is0.1%,which better meets the needs of clinical low-abundance multiple mutation detection.Overall,TTI-CS is currently one of the best methods for detecting multiple mutation detections.