Improvement of Molecular Diagnosis Using Domain‐Level Single‐Nucleotide Variants by Eliminating Unexpected Secondary Structures

Abstract

Identification of single‐nucleotide variants (SNVs) is of great significance in molecular diagnosis. The problem that should not be ignored in the identification process is that the unexpected secondary structure of the target nucleic acid may greatly affect the detection accuracy. Herein, we proposed a conditional domain‐level SNV diagnosis strategy, in which the subsequent SNV detection can only be carried out after eliminating the unexpected secondary structure of target DNA. Specifically, the target DNA is assembled into a rigid double strand, which makes folding the target DNA difficult and the unexpected secondary structure is eliminated. Based on this double‐stranded structure, specially designed probes are used to detect double‐stranded properties and report abundant domain‐level oligonucleotide information to improve the effective information in the detection results and complete domain‐level SNV diagnosis. If the unexpected secondary structure is not eliminated, the detector will first detect it and feed back to us, ensuring the accuracy of the subsequent detection results. With the occurrence (or not) of SNV and the change of the SNV site, in the proof‐of‐concept experiment, we successfully identified the four homologous sequences to be tested related to BRAF gene.

Foldback-crRNA-Enhanced CRISPR/Cas13a System (FCECas13a) Enables Direct Detection of Ultrashort sncRNA

“…Although the oligonucleotide addition system can detect ultrashort sncRNAs, adding an oligonucleotide increases the complexity of the experiment, and the detection performance is not satisfactory. Similar to the method of head-to-tail connection of crRNA and tracrRNA in the CRISPR/Cas9 system to form sgRNA, we directly synthesized complete foldback crRNA architecture (Figure A; the head-to-tail of crRNA and the oligonucleotide). − The foldback crRNA reduces reagent consumption and solves the structural instability caused by the “annealing–denaturation” dynamics at the crRNA–oligonucleotide hybrid terminal. − Further, the FCECas13a system was used for detecting target miRNA720 derivatives with different lengths, and the detection performance was compared in detail with standard Cas13a systems and oligonucleotide addition systems (Figures B and S1). Unlike the oligonucleotide addition system, the chain length of the target RNA was the major factor affecting the stability of the crRNA–target RNA hybrid in the FCECas13a system.…”

Discrimination of Multiple Homologous Sequences Based on DNA Logic Gate and Elemental Labeling Technology

“…The strategy for detection of single nucleotide variation (SNV) which can recognize a single base difference of sequences shows great potential in the detection of sequences with high homology. SNV refers to the variation of specific point on the sequence, which has been proved to be related to some diseases, including bladder cancer, 10 pancreatic cancer, 11 Alzheimer's disease 12 and cardiovascular. 13 To discriminate the SNV in the sequence, methods based on the ligase and strand displacement reaction are developed.…”