130 results about "Dna template" patented technology

Provided is a circular multimeric tandem RNA sense strand, comprising at least one sense strand sequence and at least one spacer sequence. The circular RNA is derived from an engineered parental DNA template containing all essential sequences, and sequentially comprises a first cyclization element, optionally at least one first restriction enzyme recognition sequence, at least one target sequence, optionally at least one second restriction enzyme recognition sequence, and a second cyclization element. The circular multimeric tandem RNA sense strand can bind and deliver a plurality of antisense strand RNA, increasing the binding of the sense strand and the antisense strand while utilizing the stability advantage of circular RNA.

The invention provides annular multi-tandem PCSK9 siRNA, which comprises at least one positive-sense strand sequence and at least one spacer sequence. The sense strand of the cyclic siRNA is derived from an engineered parent DNA template containing all essential sequences, comprising in the following order a first cyclization element, optionally at least one first restriction enzyme recognition sequence, at least one target sequence, optionally at least one second restriction enzyme recognition sequence, and a second cyclization element. The annular multi-tandem RNA positive-sense strand can combine and deliver a plurality of antisense strand RNAs, and the combination of the positive-sense strand and the antisense strand is increased while the stability advantage of the annular RNA is utilized.

Provided is a circular concatemeric PCSK9 siRNA, comprising at least one sense strand sequence and at least one spacer sequence. The sense strand of the circular siRNA is derived from an engineered parental DNA template containing all essential sequences, which comprises, in the following order: a first cyclization element, optionally at least one first restriction enzyme recognition sequence, at least one target sequence, optionally at least one second restriction enzyme recognition sequence, and a second cyclization element. The sense strand of the circular concatemeric RNA can bind and deliver a plurality of antisense strand RNAs, enhancing the binding between the sense and antisense strands while leveraging the stability advantage of the circular RNA.

The invention relates to the technical field of phytopathogen detection, and particularly discloses a primer, a kit and a method for detecting crop root rot fusarium solani and application, the primer comprises a specific RPA primer RPA-F and a specific RPA primer RPA-R; the specific RPA primer RPA-F is as shown in SEQ ID NO. 1, and the specific RPA primer RPA-R is as shown in SEQ ID NO. 2; the kit further comprises a guide RNA primer crRNA, and the guide RNA primer crRNA is as shown in SEQ ID NO. 3. The kit comprises a specific RPA primer RPA-F, a specific RPA primer RPA-R, a Cas12 protein, a T7 transcriptase, a crRNA DNA template, a DNA polymerase and a Cas12 / 13 nucleic acid detection test strip. The kit has the characteristics of good sensitivity, relatively good specificity and enough reliability.

The present invention relates to the field of in vitro transcription, and particularly to a plasmid backbone capable of improving the stability of polyadenylate tails. The present invention provides an expression cassette, which sequentially includes: the rop gene, the replicon ori, and the resistance gene; the rop gene, the replicon ori, and the resistance gene are connected by a linking fragment. The present invention has established a low-copy pmRVacSL and a medium-copy pmRVacM plasmid vector system through artificial modification. Since pmRVacSL is a low-copy vector, the yield of plasmid DNA is relatively low. The pmRVacM plasmid backbone solves the problem of relatively low plasmid DNA yield of pmRVacSL. This vector system can not only maintain the stable replication of polyA tail sequences of more than 150 bp, but also quickly add genes of interest and polyadenylate (polyA) sequences, and efficiently and rapidly obtain the DNA template for in vitro transcription of the target mRNA.

The present invention provides a process for the production of a closed linear DNA comprising the steps of (a) providing a DNA template comprising a DNA sequence of interest; (b) amplifying DNA from the DNA template of step (a) wherein the amplification is primed with a primase / polymerase enzyme; (c) generating a closed linear DNA with the amplified DNA produced in step (b); and (d) purifying the closed linear DNA produced in step (c). The invention also provides a closed linear DNA obtainable according to the process of the invention, a pharmaceutical composition comprising a therapeutically effective amount of the closed linear DNA of the invention, and a concatameric DNA comprising repeats of a DNA sequence of interest.

The invention provides a microsatellite genotyping method, and belongs to the technical field of genotyping. According to the microsatellite genotyping method, a bar code primer is introduced into a microsatellite primer, and the bar code primer comprises a bar code and a universal primer. The universal primer can be used for increasing PCR products obtained by the microsatellite primer so as to supplement a target product obtained by the microsatellite primer, the problem of amplification of a low-quality DNA template is solved, the library building success rate is remarkably increased to 90-100% from 60-70%, the library building and sequencing times are remarkably reduced, the sample number of each time of library building and sequencing is increased, and the library building and sequencing efficiency is improved. The method has the remarkable advantages of low cost and high accuracy.

The invention provides an ISSR-PCR reaction system for identifying kiwi fruit varieties with different colors and a method and application of the ISSR-PCR reaction system, and belongs to the technical field of molecular markers. The ISSR-PCR reaction system for identifying the kiwi fruit varieties with different colors is as follows: the DNA template is 70ng / mu L, the primer is 1.0 mu mol / L, the Mg < 2 + > is 2mM, the mix is 10mu L, and the remaining ddH2O is prepared into 20 mu L of the system. The amplification procedure is as follows: the pre-denaturation temperature is 94 DEG C, and the time is 5 minutes; the denaturation temperature is 94 DEG C for 30 seconds; the annealing temperature is set according to different primer conditions, and the time is 50s; the extension temperature is 72 DEG C, and the extension time is 1 min; circulation is performed for 35 times; and then extending at 72 DEG C for 7 minutes, and storing at 10 DEG C. The system is used for amplifying 42 kiwi fruit germplasm resources with different colors, the amplification result shows that the established ISSR-PCR reaction system is good in repeatability and high in stability, and the established ISSR molecular fingerprint spectrum can be used for variety identification.

The present application relates to the application of Klenow mutant fragments in the synthesis of nucleic acids, and belongs to the technical field of biological modification and synthesis.The present application provides the application of Klenow mutant fragments with an amino acid sequence as shown in SEQ ID NO.8 or SEQ ID NO.14 in the synthesis of nucleic acids.The mutant protein F762A expressed by the Klenow mutant fragments of the present application can efficiently synthesize deoxyribonucleic acid, ribonucleic acid, deoxyribonucleic acid analogs and ribonucleic acid analogs with different lengths of DNA templates and DNA / RNA primers at 37 DEG C, and has a wide synthesis range and universality.

The invention relates to a kit for detecting Tau-181 protein and application, and relates to the technical field of biological detection.The kit comprises a first antibody coupled with a first component of DNAzyme with catalytic activity, a second antibody coupled with a second component of DNAzyme with catalytic activity, phi29DNA polymerase, dNTPs, an annular DNA template, an initiation primer, a fluorescent beacon probe and a reaction buffer solution; the Tau-181 protein detection technology based on double-antibody sandwich coupling DNAzyme is used for quantitatively detecting the content of the Tau-181 protein in a biological sample in vitro, and can be applied to the fields of neuroscience research and clinical diagnosis, especially in the processes of early diagnosis, disease monitoring and drug research and development of neurodegenerative diseases such as Alzheimer's disease. And the method has important significance in the aspects of detection and analysis of a disease-related biomarker Tau-181 protein and the like.

The invention provides an in-vitro transcription method based on RNA polymerase and a kit, which are suitable for the fields of mRNA synthesis, gene research and biological pharmacy. According to the method, the problems of low efficiency, poor stability and the like of traditional in-vitro transcription are solved through an optimization technology. The method comprises the following specific steps: providing a linear DNA template containing a T7 promoter; according to the method, site-directed mutagenesis or fusion protein modified high-activity T7RNA polymerase is combined with chemically modified NTPs and a spermine-BSA compound stabilizer to react under the conditions that the temperature is 37-42 DEG C and the pH is 7.5-8.5, and formation of double-stranded RNA is promoted through dynamic temperature control. A reaction system contains Mg < 2 + > / Mn < 2 + > and DTT, and enzyme activity and product integrity are optimized. The kit comprises an improved reagent and a standardized process, the transcription efficiency is improved by 2-5 times, the product yield is greater than or equal to 80%, and the activity is not lost after the kit is stored at 4 DEG C for 1 month. The method has the technical advantages that NTPs degradation resistance is achieved, template preparation is simplified, universality is high, the method is suitable for large-scale production of mRNA vaccines, gene therapy and the like, the production cost is remarkably reduced, and wide application of the in-vitro transcription technology is promoted.

The invention relates to the field of DNA (Deoxyribose Nucleic Acid) sequencing, in particular to a pathogenic microorganism high-throughput sequencing detection method and a pathogenic microorganism high-throughput sequencing detection system based on multiple PCR (Polymerase Chain Reaction) detection. The method comprises the following steps: quantitatively adding an internal reference nucleic acid marker into a first microorganism sample to be detected to generate a standardized DNA template library; performing multiple PCR amplification and quality control judgment and evaluation on the standardized DNA template library to generate a PCR quality control result table; constructing a sequencing library based on the PCR quality control result table to generate sequencing library original data; performing host reference genome comparison and removal on the original data of the sequencing library, and extracting a data set to be compared; and performing pathogenic species comparative analysis according to the to-be-compared data set, and outputting a final pathogenic microorganism detection list. The invention realizes rapid and accurate detection of pathogenic microorganisms.

The invention discloses a primer, a kit and an identification method for specifically identifying sheepskin, and belongs to the technical field of leather identification. The specific primer comprises a KLHL17 forward primer as shown in SEQ ID NO. 1 and a KLHL17 reverse primer as shown in SEQ ID NO. 2. The invention also discloses a kit for detecting the specific primer. The identification method specifically comprises the following steps: 1) extracting DNA of a cortex sample to be detected to obtain a total DNA template; (2) carrying out PCR (Polymerase Chain Reaction) amplification reaction by adopting a KLHL17 forward primer, a KLHL17 reverse primer and the total DNA template to obtain an amplified DNA fragment; and (3) detecting and amplifying the DNA fragment by adopting agarose gel electrophoresis, and judging that the sample is sheep skin if the size of a PCR (Polymerase Chain Reaction) amplified band is 106bp. The method disclosed by the invention has the characteristics of rapidness, simplicity, convenience, accuracy, wide application range and low requirements on equipment, and can be used for remarkably improving the identification efficiency and reducing the detection cost. In addition, the wide application of the method is of great significance to standardize the leather market order and improve the accuracy of leather product traceability.

The invention provides a plasmid and application thereof. The plasmid comprises a recognition site of a first endonuclease, a target gene and a recognition site of a second endonuclease from a 5 '-3' direction. According to the invention, the plasmid is amplified by using a rolling circle amplification method, the rolling circle amplification product is cleaved by using the first endonuclease and the second endonuclease, the enzyme cleaved product is digested by using exonuclease, and the product is further purified by using oligonucleotide. The obtained product can be used for in vitro transcription or directly applied to eukaryotic expression. According to the scheme provided by the invention, a simple, high-yield, economic, rapid and amplifiable preparation process of the full chemical synthesis DNA template for escherichia coli fermentation is established.

Provided are a circular functional nucleic acid, a preparation method therefor and a use thereof. The method involves combining a functional nucleic acid with a circular nucleic acid, using a method combining DNA template-directed assembly with enzymatic conjugation to first prepare a single-stranded circular DNA, synthesize a corresponding outer short strand, and conjugate a functional nucleic acid drug onto the outer short strand, and optimizing and screening reaction conditions, thereby preparing a circular functional nucleic acid. The circular functional nucleic acid has the characteristics of adjustable size, adjustable valence state (multivalent or polyvalent), high stability, and the capability for chemical conjugation with drugs or drug incorporation, etc.; a novel circular multivalent functional nucleic acid technology is created, which significantly enhances the targeting ability and stability of nucleic acid drugs; thus, a nucleic acid drug development platform o-FLARE having efficient targeting, immune agonism, and protein degradation functions can be established.

A method and composition for preparing a DNA library for replicating a target nucleic acid sequence are provided. A target DNA template containing the target sequence is circularized via a terminal adapter to form a circular construct, which is extended bidirectionally by polymerase-mediated elongation initiated at a nick site on the terminal adapter. Following polymerase-mediated elongation, a diploid DNA template is formed containing two copies of the target DNA template (and thus two copies of the target sequence). Each strand of the diploid DNA template contains a parent polynucleotide chain ligated to a newly synthesized daughter strand copy of the parent polynucleotide chain. Predetermined sequences, such as primer sequences, unique molecular identifiers, and sequence indices, can be included in the diploid DNA template. Sequencing of the diploid DNA template can reveal genetic / epigenetic information related to the target sequence. A method for constructing asymmetric and multiploid DNA template constructs is also provided.

The application discloses a primer, a kit and an identification method for identifying a leather cultural relic species, and belongs to the field of leather cultural relic molecular archaeology. The application provides an upstream primer cytbF shown as SEQ ID NO. 1 and a downstream primer cytbR shown as SEQ ID NO. 2. The specific method is as follows: (1) extracting DNA of a leather sample to be detected to obtain a total DNA template; (2) performing PCR amplification reaction on the total DNA template by using the upstream primer cytbF and the downstream primer cytbR to obtain an amplification product; (3) performing bidirectional sequencing or second-generation sequencing on the amplification product, and performing BLAST similarity comparison on the sequencing result to determine the species of the leather sample to be detected. The primer can be used not only for amplification of single leather sample DNA, but also for mixed leather samples, and in addition, can be used for identification of leather samples damaged by acid and alkali in the environment. The identification method provided by the application is rapid, simple and accurate, and has important significance in the field of cultural relic identification, protection and restoration.

The present invention relates to a bioreactor for RNA in vitro transcription, a method for RNA in vitro transcription, a module for transcribing DNA into RNA and an automated apparatus for RNA manufacturing. Further, the use of a bioreactor for RNA in vitro transcription as described herein is part of the present invention. The present invention relates to an RNA in vitro transcription reactor designed to be operable in an automated manner under GMP-compliant conditions. In particular, said RNA in vitro transcription reactor allows repetitive use of DNA template for various RNA in vitro transcription reactions. Further, the invention relates to an apparatus for RNA manufacturing comprising (a) a module for template DNA synthesis, (b) a module for transcribing DNA into RNA comprising said RNA in vitro transcription reactor, and, optionally, (c) a module for RNA formulation.

Provided are DNA library preparation methods and compositions that duplicate a target nucleic acid sequence. A target DNA template including the target sequence is circularized via an end adapter to form a circular construct, which is bidirectionally extended by a polymerase-mediated extension that is initiated at nick sites of the end adapter. Following polymerase-mediated extension, a double-length DNA template is formed that includes two copies of the target DNA template (and hence two copies of the target sequence). Each strand of the double-length DNA template includes a parental polynucleotide strand joined to a newly synthesized daughter strand copy of the parental polynucleotide strand. Predetermined sequences can be included in the double-length DNA template, such a primer sequences, unique molecule identifiers, and sequence indexes. Sequencing of the double-length DNA template can reveal genetic / epigenetic information associated with the target sequence. Also provided are methods to create asymmetric and multi-length DNA template constructs.

The present invention discloses a capillary monolithic column modified with a deoxyribozyme nanoflower, a preparation method thereof, and an application thereof in separating chiral molecules, belonging to the field of pharmaceutical analysis. The present invention synthesizes a deoxyribozyme-based nanoflower chiral selector, the nucleotide sequence of the single-stranded DNA template of the deoxyribozyme being shown in SEQ ID NO.1, and the synthesized deoxyribozyme nanoflower having good pH and temperature stability. The prepared deoxyribozyme nanoflower is bonded to a graphene oxide-functionalized capillary silica monolithic column, and chiral molecules are separated under electrophoretic conditions, achieving baseline separation of 2'-deoxyadenosine, atenolol, propranolol, tyrosine, and nafopam enantiomers.

The invention discloses a two-color fluorescent aptamer sensor for detecting kanamycin and aflatoxin B1 in feed by using two-color fluorescent silver nanoclusters, and belongs to the field of food safety detection. According to the invention, different DNA templates are utilized to synthesize the double-color silver nano-cluster as a fluorescent probe, and the double-color silver nano-cluster has two different fluorescent colors of 530 nm and 630 nm. And a sequence rich in guanine is used as a capture probe, so that an amplification effect on signals of two kinds of fluorescence is achieved. When KAN or AFB1 exists, a corresponding target object is selectively combined with an aptamer fragment to form an aptamer-target object-aptamer sandwich structure, and the fluorescence intensity of the silver nano-cluster is induced to be enhanced through G-rich sequence and base pairing. The'lightening 'type double-color fluorescent aptamer sensor shows excellent detection performance, the KAN detection limit reaches 0.86 nM, the AFB1 detection limit reaches 0.71 nM, the'lightening' type double-color fluorescent aptamer sensor can be used for detecting kanamycin and aflatoxin B1 in a feed sample, and the reliability and application potential of the'lightening 'type double-color fluorescent aptamer sensor in multiple detection of antibiotics and toxins in the feed sample are highlighted.

The invention belongs to the technical field of biology, and particularly relates to a DNA polymerase mutant for improving amplification efficiency of a high GC sample and application of the DNA polymerase mutant. According to the invention, DNA polymerase mutants KODG and KODC are obtained through site-specific mutagenesis, and on the basis of retaining the capability of amplifying a DNA template with conventional GC content, the capacity of amplifying a DNA template with high GC content (gt; compared with the prior art, the method has the advantages that the amplification efficiency of a DNA template (3kb, 60%) is improved, effective amplification of difficult-to-amplify fragments with 3kb, 95% GC content and the like is successfully realized, and the technical bottleneck of amplification failure caused by high thermal stability and difficulty in complete melting of the existing polymerase in a high-GC region is overcome, so that the application range of the DNA polymerase in complex template amplification is expanded, and the application prospect is broad. And a more reliable enzymology tool is provided for related detection and identification of high-GC-content samples.

A method for transcribing a DNA template into RNA in vitro includes providing a mixture comprising a buffer material, ribonucleoside triphosphate (NTP), one or more magnesium salts at a concentration of from about 2 mM to about 60 mM, the DNA template, and a recombinant RNA polymerase, and incubating the reaction mixture at from about 25 DEG C to about 40 DEG C for from about 1 hour to about 12 hours, thereby producing the RNA. In one embodiment, a method for in vitro transcription includes providing a DNA template and a cap analog that binds-1 and / or + 1 nucleotides of a promoter for in vitro transcription, thereby producing mRNA of more complete length, allowing for more flexible selection of a first mRNA base, and providing a + 2 position open to a custom sequence.

The invention discloses a QPCR (Quantitative Polymerase Chain Reaction)-based DNA (Deoxyribose Nucleic Acid) methylation rate detection method, which comprises the following steps of: treating a DNA sample to be detected by bisulfite, dividing the DNA sample into a first sample and a second sample which are parallel to each other, and performing substrate amplification on a methylated DNA template and a non-methylated DNA template in the samples through PCR amplification by adopting the same amplification primer under the same condition; then respectively depleting dATP in a sample A reaction system and dGTP in a sample B reaction system through a template and a primer with thymine deoxyribonucleotide repetitive sequences or a template and a primer with cytosine deoxyribonucleotide repetitive sequences, and controlling independent detection of methylation and non-methylation templates through a d3TP system lacking dATP or dGTP; and finally, directly analyzing the fluorescence signal of the methylation template and the fluorescence signal of the non-methylation template, and calculating the methylation rate of the DNA sample to be detected through a methylation rate calculation function.

This invention discloses a CRISPR / Cas-regulated DNA silver nanocluster sensor and its method and application for detecting adulteration in meat, belonging to the field of food safety testing technology. This DNA-templated silver nanocluster sensor introduces a novel tagless fluorescent probe, DNA-AgNCs, as a substrate for CRISPR / Cas12a. After the CRISPR / Cas12a-crRNA complex specifically recognizes the target nucleic acid, its trans-cleavage activity is activated, leading to the cleavage of the DNA template and a significant decrease in fluorescence signal. Detecting adulteration in meat using this DNA-templated silver nanocluster sensor can cleverly convert the concentration of adulteration into a quantifiable fluorescence signal output, achieving quantitative detection of adulterants. It exhibits high sensitivity and specificity, providing a universal and sensitive detection method for meat adulteration.

The invention discloses a hot-start high-specificity LAMP (loop-mediated isothermal amplification) isothermal nucleic acid rapid detection method which comprises the following operation steps: step 1, preparing three pairs of LAMP amplification primers, the 3'ends of one pair of primers F3 and B3 being one of ddC / ddG / ddT / ddA or other closed groups, and each of the primers F3 and B3 internally containing an RNA (ribonucleic acid) basic group; 2, establishing an LAMP isothermal nucleic acid amplification reaction system by using three pairs of primers; 3, in the process of hot start of the high-specificity LAMP amplification reaction, after the primers F3 and B3 are paired with the template DNA, cutting off the DNA at the RNA base by thermal stability endonuclease HII to generate a double-dumbbell initiator (a product 6 in the figure 1), thereby starting the LAMP continuous amplification reaction; if the primers F3 and B3 cannot be specifically paired with the DNA template, the primers F3 and B3 cannot be cut by thermal stability endonuclease HII, and a double-dumbbell type initiator cannot be generated, so that the LAMP continuous amplification reaction cannot be started.

In one aspect, the disclosure relates to a system for site-specific modification of a double­stranded target DNA sequence, the system including at least: a fusion protein comprising an RNA-binding nickase protein and a DNA-dependent DNA polymerase, and a chimeric guide nucleic acid sequence comprising a guide RNA sequence that interacts with the RNA-binding nickase protein and a single-stranded DNA template, wherein the single-stranded DNA template comprises a modified sequence for insertion into a second strand of the double-stranded target DNA sequence and a primer binding site. Also disclosed is a method for site-specific modification of a double-stranded target DNA sequence in a cell. In another aspect, the present disclosure is directed to a fusion protein including an RNA-binding nickase protein and a DNA-dependent DNA polymerase, wherein one or both of the nicakse protein and the DNA polymerase can include one or more mutations to optimize function and prevent off-target effects.

The invention discloses a hairpin type DNA-copper cluster probe for rapid ratio fluorescence detection of Hg < 2 + > and a preparation method, the hairpin type DNA-copper cluster probe contains a hairpin type DNA template (hpDNA) capable of emitting strong red fluorescence and weak blue fluorescence at the same time, and the sequence of the hairpin type DNA template is 5 '-TTTTTTTTTTTTTTTTTTTTT TTTTCGCGCGATATATCGCGCG-3'. The method comprises the following steps: preparing a single-stranded DNA aqueous solution by using a 3-morpholine propanesulfonic acid buffer solution, heating the single-stranded DNA aqueous solution in a constant-temperature water bath kettle, cooling to room temperature, and refrigerating and storing in a refrigerator to form an hpDNA template solution; adding a copper sulfate solution into the hpDNA template solution, and incubating at constant temperature to obtain a mixed solution; and adding a sodium ascorbate (SA) solution into the mixed solution, reacting at a constant temperature, and reducing Cu < 2 + > coordinated on the hpDNA template by sodium ascorbate to obtain the hairpin type DNA-copper cluster probe for rapid ratio fluorescence detection of Hg < 2 + >. The hairpin DNA-copper cluster (hpDNA-CuNCs) probe can be used for rapid ratio fluorescence detection of Hg < 2 + >. The hpDNA-CuNCs probe has excellent selectivity on Hg < 2 + >, is used for rapid and sensitive detection of trace Hg < 2 + > in an actual water body sample, and has an accurate detection result.

The present invention relates to the field of biomedicine technology, and specifically discloses a circular mRNA vaccine for marine farmed fish, a preparation method thereof, and an application thereof. The circular mRNA in the present invention includes an IRES untranslated region, a signal peptide sequence, and an NNV virus antigen coding region connected in sequence; in the preparation process, a DNA template with a T7 promoter is first synthesized by a chemical method, and T7 RNA transcription is performed using the DNA template. The transcribed RNA is adenylated, and then circularized and connected by T4 RNA enzyme 2 to generate circular RNA, and finally purified by ethanol precipitation to obtain NNV-B1 circular mRNA. The NNV-B1 circular mRNA vaccine prepared by the present invention has been shown by the results of the animal safety test to be safer and more effective, convenient and simple to use clinically, and has broad application prospects in the prevention and control of viral neuronecrosis.

An embodiment of the present invention relates to a method, apparatus, device, and storage medium for estimating sequencing depth in dual sequencing. The method includes: assigning tags to multiple DNA templates in the same proportion according to the proportion of double-stranded templates, forward single-stranded templates, and reverse single-stranded templates, and assigning a template ID to each of the DNA templates; generating saturated sequencing data; generating mutant templates and mutant-supported reads; setting the sub-sampled reads to a specified sequencing depth and then statistically analyzing the mutant detection frequency; repeating the steps of generating saturated sequencing data, generating mutant templates and mutant-supported reads, and statistically analyzing the mutant detection frequency after setting the sub-sampling multiple times, taking the average value of the mutant detection frequencies as an estimated value of the detection probability at the specified sequencing depth, and the sequencing depth corresponding to when this value reaches a set threshold is the sequencing depth required to stably detect mutants. The technical solution of the embodiment of the present invention can recommend the sequencing depth that should be used to ensure stable detection of mutants during dual sequencing.