A method and a kit for detecting genome editing and application thereof belongs to the field of genome editing efficiency detection, and the getPCR method for determining genome editing efficiency includes quantifying wild-type DNA in a genome to be tested and calculating the percentage of the wild-type DNA to determine the genome editing efficiency. The method has been proved to have good detection accuracy and simple operation, and can be applied to all genome editing methods to quantify genome editing efficiency and screen single-cell clones.

Provided herein are systems and methods for detecting genomic structural variants using a non-application gene-editing sample preparation followed by long-read sequencing.

There is described herein, a method of capturing and analyzing cell-free methylated DNA in a sample. The method involves subjecting the sample to library preparation to permit subsequent sequencing of the cell-free methylated DNA. A predetermined amount of control synthetic DNA fragments are added to the sample. The control synthetic DNA fragments each have a known nucleic acid sequence that does not align to a target genome sequence, and at least some of the control synthetic DNA fragments are methylated. The sample is denatured, and cell-free methylated DNA and the control synthetic DNA fragments are captured using a binder selective for methylated polynucleotides. The captured DNA is amplified and sequenced.

There is described herein, a method of capturing and analyzing cell-free methylated DNA in a sample. The method involves subjecting the sample to library preparation to permit subsequent sequencing of the cell-free methylated DNA. A predetermined amount of control synthetic DNA fragments are added to the sample. The control synthetic DNA fragments each have a known nucleic acid sequence that does not align to a target genome sequence, and at least some of the control synthetic DNA fragments are methylated. The sample is denatured, and cell-free methylated DNA and the control synthetic DNA fragments are captured using a binder selective for methylated polynucleotides. The captured DNA is amplified and sequenced.

Aspects of the present disclosure relate to methods for detecting mutant TERT promoter sequences (e.g., C228T, C250T) that provide several improvements over conventional detection methods, thereby enabling detection of such mutations in biological fluid samples (e.g., plasma), which contain miniscule amounts of nucleic acids. Such improvements include, but are not limited to, improvements in detection sensitivity and specificity, which allows detection of mutations in a biological sample having a low level of nucleic acids such as a plasma sample from a patient having brain cancer (e.g., glioma).

A nucleic acid testing device includes: a stage on which is placed a tissue section to which a solution has been added, in which the solution contains a labeling substance of a target nucleic acid and an amplification reagent for the target nucleic acid; a temperature adjuster that adjusts the temperature of the tissue section on the stage; a temperature controller that controls the temperature adjuster to advance nucleic acid amplification reaction in the tissue section; an intensity detector that detects label intensity in the tissue section over time; and a storage unit that stores detection information generated by the intensity detector.

A nucleic acid testing device includes: a stage on which is placed a tissue section to which a solution has been added, in which the solution contains a labeling substance of a target nucleic acid and an amplification reagent for the target nucleic acid; a temperature adjuster that adjusts the temperature of the tissue section on the stage; a temperature controller that controls the temperature adjuster to advance nucleic acid amplification reaction in the tissue section; an intensity detector that detects label intensity in the tissue section over time; and a storage unit that stores detection information generated by the intensity detector.

An object of the present invention is to provide a novel method for designing a primer ensuring reactivity and discriminatory power in a method for detecting a single base substitution based on an ASP-PCR method and to provide a method for easily detecting multiple point mutations within overlapping amplicons, particularly, two adjacent single base substitutions. The single base substitutions can easily be detected by using a mutant primer in which the base of the third nucleotide from the 3′ end corresponds to the base of a mutant nucleotide of a single base substitution contained in a nucleic acid sample, in which the base of the second nucleotide from the 3′ end is not complementary to the base of the corresponding nucleotide of the nucleic acid, and in which the bases of the other nucleotides are complementary to the bases of the corresponding nucleotides of the nucleic acid.

An object of the present invention is to provide a novel method for designing a primer ensuring reactivity and discriminatory power in a method for detecting a single base substitution based on an ASP-PCR method and to provide a method for easily detecting multiple point mutations within overlapping amplicons, particularly, two adjacent single base substitutions. The single base substitutions can easily be detected by using a mutant primer in which the base of the third nucleotide from the 3′ end corresponds to the base of a mutant nucleotide of a single base substitution contained in a nucleic acid sample, in which the base of the second nucleotide from the 3′ end is not complementary to the base of the corresponding nucleotide of the nucleic acid, and in which the bases of the other nucleotides are complementary to the bases of the corresponding nucleotides of the nucleic acid.

The present invention belongs to the field of diagnosis of disease. Thus the present invention is focused on a method and kit and system for quantifying the level of minimal residual disease (MRD) in a subject who has been treated for said disease, as well as a method of treatment of said disease in a subject which comprises a step of quantifying the level of minimal residual diseases, wherein said quantifying comprises: (a) identifying, amplifying and sequencing a nucleotide sequence in a biological sample obtained from said subject after treatment for said disease, wherein the gDNA of said biological sample has an average weight, k, per cell, and wherein said nucleotide sequence is identified using primers and is amplified using an amount, D, to afford a first list of characters; (b) identifying, amplifying and sequencing a nucleotide sequence in a biological sample obtained from a subject with said disease using the same primers as in step (a) to afford a second list of characters; (c) determining, for each first list of characters obtained in step (a), the degree of similarity, DS, with each second list of characters obtained in step (b); (d) selecting, for each first list of characters obtained in step (a), the DS of highest value, DS.sub.HV; (e) adding up the number of first lists of characters obtained in step (a) which have a DS.sub.HV that is greater than a threshold value, T, to obtain L.sub.c; (f) adding up the total number of lists of characters, L.sub.t, in the first list of characters; and (g) calculating the level of minimal residual disease (MRD) according to either of the following formulae:
MRD=(L.sub.c×k)/(L.sub.t×D)
or
MRD=L.sub.c/L.sub.t
or
MRD=L.sub.c×(D/k)/L.sub.t.sup.2.