72 results about "Amplification/Sequencing" patented technology

The invention belongs to the technical field of molecular biology, and particularly relates to a single-copy nuclear gene marker of elaeagnus mollis, a primer pair and application of the single-copy nuclear gene marker. The elaeagnus mollis single-copy nuclear gene marker comprises any one or more of elaeagnus mollis single-copy nuclear gene markers 1, 2, 3, 4, 5, 6 and 7; the nucleotide sequences of the single-copy nuclear gene marker of the elaeagnus mollis are sequentially as shown in SEQ ID NO.1 to SEQ ID NO.7. The invention further discloses a preparation method of the single-copy nuclear gene marker. The single-copy nuclear gene marker primer pair of elaeagnus mollis provided by the invention has very high universality, amplification sequencing can be performed in selected elaeagnus mollis individuals of different provenances, a genetic diversity index analysis result is good, and all individuals of different provenances can be obviously distinguished according to a genetic structure analysis result; the method is suitable for genetic diversity analysis of large-scale germplasm resources of elaeagnus mollis.

The invention discloses a method for constructing an Asiatic corn borer GBP gene homozygous mutant, which comprises the following steps: amplifying and cloning an Asiatic corn borer GBP gene sequence by using RACE technology, and the nucleotide sequence of the Asiatic corn borer GBP gene is as shown in SEQ ID No.1; two sgRNA target sequences are designed on a second exon of a GBP gene sequence of the ostrinia furnacalis, target sgRNA is synthesized through in-vitro transcription, the target sgRNA and Cas9 protein are fused in vitro to form an RNP mixture, and the RNP mixture is injected into ovum of the ostrinia furnacalis; the method comprises the following steps: carrying out PCR (Polymerase Chain Reaction) amplification on a target spot by using a target spot detection primer, sequencing to confirm a mutant individual, hybridizing and selfing to obtain a GBP homozygous mutant population. The Asiatic corn borer GBP gene homozygous mutant is constructed, so that the reproduction and immune functions of the mutant are remarkably reduced, and reference and research basis are provided for later development and formation of efficient pest biopesticide.

A method for preparing a polynucleotide sample for sequencing is disclosed. The method involves preparing a fractionated amplified sequencing library in which each polynucleotide comprises a first indexing barcode at a first end but not a second end.

The present invention relates to aqueous compositions, and more specifically aqueous compositions for replicating a nucleic acid template, wherein the composition comprises D2O, as well as to methods and uses relating to such compositions. The composition can be used, for example, in a method of storing the composition, or a method for replicating a nucleic acid template, for instance for the synthesis of Xpandomers for sequencing by expansion.

The invention relates to a method for identifying Langya chicken and Non-Langya chicken by using SNP molecular markers, and belongs to the technical fields of animal husbandry and molecular biology. The SNP molecular marker for Langya chicken identification comprises 12 loci. The invention further provides a primer combination which comprises SNP loci for amplifying Langya chicken identification. According to the method, the 12 specific SNP sites of the Langya chicken are utilized, the site information is obtained through PCR amplification and Sanger sequencing, prediction is conducted through machine learning, the Langya chicken variety identification model is established based on the 12 SNP site combination, and the method has the advantages of being low in cost, simple in sequencing method, high in speed and high in accuracy.

The application discloses a kind of sheep mycoplasma Mo, Mccp, Mmc triple PCR detection method establishment and application, by using high-throughput Mauve genome collinearity analysis the specific difference section between three kinds of sheep mycoplasma genomes each other, design and screen out the specific primer capable of differential diagnosis three kinds of sheep mycoplasma, by primer sequence amplification, sequencing, identify three new sheep mycoplasma differential diagnosis target molecule, respectively Mo differential diagnosis target molecule DprA, Mccp differential diagnosis target molecule MCCPF38_00240, Mmc differential diagnosis target molecule restriction endonuclease subunit S.Utilize new target specific section, establish the triple PCR method capable of simultaneous differential diagnosis three kinds of sheep mycoplasma (Mo, Mccp, Mmc).The PCR method of the application has the characteristics of clinical convenient, rapid diagnosis, can be widely used in the clinical diagnosis of sheep mycoplasma, with good market prospect and economic value.

Provided is a high-resolution promoter interaction fragment targeted capture method suitable for plants, which comprises: constructing a chromatin conformation capture pre-library; designing an RNA probe reversely complementary to a core promoter sequence; using the RNA probe to capture a promoter interaction fragment in the chromatin conformation capture pre-library; amplifying the library; and performing sequencing to obtain information of the promoter interaction fragment. By means of complementarily pairing hybridization between an RNA and a DNA, the capture method can specifically capture a promoter interaction fragment from a high-resolution chromatin conformation capture library, so as to obtain information of the promoter interaction fragment. Compared with existing capture technologies, the method exhibits greatly improved capture efficiency and has an obvious advantage in searching for distal regulatory elements.

The invention discloses a preparation method of a space chip, the space chip and application of the space chip, the preparation method comprises the following steps: hybridizing: connecting a single-stranded nucleic acid molecule to the surface of a solid-phase substrate, the single-stranded nucleic acid molecule comprises a first fixed sequence, a space bar code sequence, a sequencing primer binding sequence and a second fixed sequence, the surface of the solid-phase substrate of the chip substrate is connected with a first probe, and the first probe comprises a first fixed sequence complementary sequence and a second fixed sequence complementary sequence; carrying out ring closing to enable the single-stranded nucleic acid molecules to form annular nucleic acid molecules; carrying out rolling circle amplification; sequencing to obtain sequence information of the space bar code sequence; synthesizing single-stranded nucleic acid molecules by taking the rolling circle amplification product as a template to obtain a single-stranded nucleic acid molecule library; fixing and clustering: fixing the single-stranded nucleic acid molecule library on the surface of a solid-phase substrate to form a single-stranded nucleic acid molecule cluster. According to the invention, the chip with the nucleic acid probe carrying spatial position coordinate information can be prepared.

The application provides a molecular marker related to sheep growth traits and application thereof, the molecular marker is designed by primers according to the sequence of the LGALS3 gene, DNA is extracted from sheep blood, and through PCR amplification, DNA sequencing and sequence analysis, it is found that a T / C polymorphism site exists at the 298th position of the amplified fragment, further, the polymorphism site of 849 Hu sheep is detected by using AQP primers, a least square model is established, genotype and growth traits are associated, finally, it is determined that Y at the 298th bp of the amplified LGALS3 gene fragment shown in SEQ ID NO. 1 is T or C, the mutation leads to T / C polymorphism of the molecular marker, and the molecular marker can be used as a molecular marker related to sheep growth traits. The molecular marker of the application can be used for screening a new variety of fast-growing high-quality mutton sheep, and can be used for screening assisted breeding, cultivating fast-growing sheep, and helping to increase economic benefits.

The invention provides a full-process automatic all-in-one machine for DNA extraction, amplification and sequencing. The full-process automatic all-in-one machine comprises a cabin body, the cabin body is internally provided with a first space, and the first space is internally provided with a biological sample extraction workbench for realizing the whole process of DNA extraction, amplification and sequencing; the top of the cabin is provided with an air inlet duct, the side surface or / and the bottom of the cabin is provided with an air outlet duct, and the air inlet duct and the air outlet duct form negative pressure in the cabin; an amplification area is arranged on the biological sample extraction workbench, a cooling base used for reducing pollutants generated in the amplification process is arranged in the amplification area, and an air suction plate is arranged on the side face of the amplification area; the air suction plate is communicated with the air outlet channel, and pollutants generated in the amplification area are discharged out of the cabin body through the air outlet channel. Pollutants generated in the cabin body can be effectively prevented from facing three positions outside from gaps or cabin door lamps of the cabin body, so that pollution to the surrounding environment is avoided, and the safety of biological experiments is further improved. In addition, the integrated cabin is provided, and the requirement for the operation space of the biological experiment is effectively reduced.

This invention provides a gene related to loquat flowering. SVP1 This invention relates to the field of molecular biology, specifically to a tightly linked SNP marker and its applications. The nucleotide sequence of this SNP marker is shown in SEQ ID NO.1. A genotype of C at the locus corresponds to a spring-flowering phenotype in loquat, while a genotype of G corresponds to an autumn-flowering phenotype. The invention also provides specific primers (SEQ ID NO.2 and SEQ ID NO.3) for detecting this SNP marker, a kit containing these primers, and a screening method for detecting loquat flowering time using this marker. By extracting loquat DNA and performing PCR amplification and sequencing analysis, the flowering period of loquat can be quickly and accurately determined. The SNP marker of this invention is highly linked to loquat flowering time, providing an effective tool for marker-assisted selection breeding of loquat at different flowering periods. This significantly improves breeding efficiency, shortens the breeding cycle, and is of great significance for extending the loquat fruit supply period and optimizing the industrial structure.

The invention discloses an environmental DNA species monitoring method based on Chinese sturgeons, and relates to the technical field of biological gene detection.According to the environmental DNA species monitoring method based on the Chinese sturgeons, mitochondrial whole genomes of the Chinese sturgeons are determined through a method combining a second-generation library-building sequencing technology and a third-generation library-building sequencing technology, and the whole genomes of the mitochondria of the Chinese sturgeons are determined through probe design and PCR amplification sequencing. The sequence of the whole mitochondrial genome of the Chinese sturgeons is combined for comparison, and the specificity difference between the Chinese sturgeons and other related sturgeons is distinguished, so that the efficiency of detecting rare and endangered species Chinese sturgeons by an environmental DNA method is improved, and the accuracy of species identification is improved.

The invention provides a method suitable for high-resolution targeted capture of promoter interaction fragments of plants. The method comprises the following steps: constructing a chromatin conformation capture pre-library; designing an RNA probe which is reversely complementary with the core promoter sequence; capturing a promoter interaction fragment in the chromatin conformation capture pre-library by using an RNA probe; performing library amplification; and sequencing to obtain promoter interaction fragment information. According to the capturing method, the promoter interaction fragment is specifically captured from the high-resolution chromatin conformation capturing library through complementary pairing hybridization of RNA and DNA, and then promoter interaction fragment information is obtained. And compared with the existing capture technology, the capture efficiency is greatly improved, and the method has obvious advantages in searching far-end regulation and control elements.

Some embodiments relate to methods, systems, uses, or software for identification of structural variants (SVs) for regions of deoxynucleic acid (DNA) sequences. A device may receive, at a processor, a dataset comprising base pair data output from a sequencing by expansion process. A device may extract, at the processor, soft-clipped reads from the received dataset. A device may group, at the processor, the extracted soft-clipped reads based at least on their respective breakpoint. A device may determine, at the processor, a presence or an absence of a somatic structural variant by k-mer matching the grouped and extracted soft-clipped reads. A device may provide, at the processor, at least one determined somatic structural variant responsive to determining the presence of the somatic structural variant

The invention belongs to the field of fish species and sex identification in the field of aquaculture, and discloses a specific DNA fragment combination for distinguishing female acipenser ussuriensis and female Huso dauricus and application. According to the invention, a segment of specific DNA fragment for distinguishing genders of female acipenser ussuriensis and female Huso dauricus is successfully screened through conventional PCR amplification, sequencing and sequence comparative analysis, primers are designed to accurately and rapidly identify genders of female acipenser ussuriensis and female Huso dauricus, and genders and female parent sources of female acipenser ussuriensis and female Huso dauricus individuals and hybrid generations thereof are distinguished. The invention also discloses a primer sequence, a PCR reaction system and conditions. The identification method disclosed by the invention is simple, rapid and accurate to operate, has extremely small harm to fish bodies, solves the problems of identifying the genders of the Russian sturgeon and the Huso dauricus, the female parent of the hybrid sturgeon and the caviar source, and is beneficial to the development of the sturgeon caviar industry.

The present application belongs to the field of molecular biology and animal molecular breeding, and discloses a method for identifying genetic composition of hybrid offspring of Misgurnus anguillicaudatus and Culter alburnus, a specific primer pool, and application of the specific primer pool in identifying genetic composition of hybrid offspring of Misgurnus anguillicaudatus and Culter alburnus. The present application innovatively uses a method combining multiplex PCR and high-throughput sequencing to qualitatively and quantitatively detect base conditions of SNP sites on each pair of homologous chromosomes of Misgurnus anguillicaudatus and Culter alburnus, thereby providing an efficient identification technology for genetic composition of hybrid offspring obtained between Misgurnus anguillicaudatus and other species of close relatives such as Culter alburnus. Compared with whole genome sequencing genes, the method only amplifies and sequences specific regions on each chromosome, which effectively reduces sequencing cost and analysis period, and is an efficient technology for rapidly identifying genetic composition of hybrid offspring obtained by hybridization of Misgurnus anguillicaudatus and other close relatives such as Culter alburnus.

The present invention relates to methods and compositions for preparing a sample enriched for single stranded nucleic acids. The single stranded nucleic acids find use as templates for synthesizing Xpandomer copies of the nucleic acids. The Xpandomer copies may be passed through a nanopore sensor to determine the nucleic acid sequence of the nucleic acids. The methods include asymmetric PCR, amplicon immobilization melt, exonuclease-mediated partial or complete digestion of one or both strand of an amplicon and linear amplification.

The invention discloses an SNP (Single Nucleotide Polymorphism) marker related to chicken body size traits and application of the SNP marker in assistant breeding of Lin chickens, and belongs to the field of molecular genetics and gene breeding. The SNP marker is located at 172801329bp (reference genome GRCg7b) of a No.1 chromosome of the Lin chicken, corresponds to the 201st site of SEQ ID No.1 and has T / A polymorphism, and the phenotypic values of eight body size characters of AA genotype individuals are remarkably higher than those of AT and TT genotypes. Meanwhile, the invention provides a specific primer group as shown in SEQ ID No.2-3 and an auxiliary breeding method of the specific primer group. The genotype is determined through DNA extraction, PCR amplification and sequencing, and AA genotype individuals are screened. The method solves the problems that traditional breeding is long in period, accuracy is interfered and the like, early-stage accurate breeding is achieved, the method can be used for preparing a detection kit, and technical support is provided for improvement of the Lin-meadow chicken variety.

The invention relates to the technical field of gene detection, in particular to an opioid analgesic drug gene detection method based on multiple targeted amplification, which comprises the following steps: extracting DNA from a peripheral blood sample; the method comprises the following steps: setting a primer pool aiming at a preset site of a specific gene related to opioid drugs, and carrying out multi-targeted amplification sequencing by using the primer pool to obtain sequencing data; comparing the sequencing data with a human reference genome to obtain a data comparison rate, and judging the eligibility of the multi-target amplification sequencing process according to the data comparison rate; performing variation detection on the qualified sequencing data, identifying the base type of each SNP site, and determining the genotype of the subject in combination with a dbSNP database; and inputting the genotype and clinical data into a pre-trained multi-gene prediction model, and obtaining the medication guidance of the subject for using the opioid drugs. The gene detection efficiency is improved.

Provided herein are methods of targeted genomic sequencing requiring only a single amplification step. The Single Amplification Sequencing (SAS) assay provided herein amplies and sequences targeted genomic regions at reduced time and cost. The assay provides robust coverage and accurate drug resistance profiles for multiple first- and second-line Tuberculosis drugs, showcasing its clinical utility for rapid antimicrobial resistance (AMR) detection. SAS significantly simplifies targeted Next Generation Sequencing (tNGS) workflows and holds significant promise for revolutionizing precision diagnostics across a wide array of clinical and research settings.

The present invention relates to methods and compositions for preparing a sample enriched for single stranded nucleic acids. The single stranded nucleic acids find use as templates for synthesizing Xpandomer copies of the nucleic acids. The Xpandomer copies may be passed through a nanopore sensor to determine the nucleic acid sequence of the nucleic acids. The methods include asymmetric PCR, amplicon immobilization melt, exonuclease-mediated partial or complete digestion of one or both strand of an amplicon and linear amplification.

The invention provides a kit for HLA-B27 allele typing detection. The kit comprises an amplification primer covering an HLA-B exon region, and a PCR (Polymerase Chain Reaction) reaction solution. According to the detection method, 1 / 2, 3 and 4 exons of HLA-B27 are covered, DNA samples are amplified and sequenced through a special primer group, HLA-B locus alleles are obtained through database comparison, interference of other homologous high locus gene sequences is avoided, and the accuracy of typing results is improved. According to the method, the inherent defects of a traditional method in the aspects of coverage range, anti-interference capability, operation convenience and the like are overcome, innovative improvement is achieved in multiple technical levels, an efficient and reliable tool is provided for early screening of ankylosing spondylitis, subtype correlation research and individualized treatment, and the method is suitable for popularization and application. The detection accuracy, sensitivity and practicability are obviously improved.

The invention discloses a construction method and application of a single-cell complete transcriptome library. The method comprises the steps that a universal primer is used as a cDNA synthesis primer and template conversion oligonucleotide at the same time, an amplifiable cDNA product is synthesized through a reverse transcription reaction, the universal primer is composed of a fixed sequence composed of three basic groups G, A and T, and the length of the universal primer is 10-30 nt; at least one thymine in the fixed sequence of the universal primer is replaced by deoxyuracil; the universal primer is partially degraded through special modification on the universal primer by utilizing a specific enzyme comprising reverse transcriptase MMLV with template conversion activity and a mutant of the reverse transcriptase MMLV, or TGIRT-III and a mutant of the TGIRT-III; a cDNA amplification product with chain specificity is obtained by using a specific amplification primer and method. By utilizing the characteristic that a universal primer is randomly and efficiently combined with RNA molecules at low temperature, a barcode is added to a single cell in combination with a specific coding microsphere, so that high-sensitivity and whole transcriptome amplification sequencing of the high-throughput single cell is realized.

The disclosure relates to a classification method and device, electronic equipment and storage medium, the method comprising: extracting the number of amplicons of various methylation patterns in each target amplification region and the number of amplicons of each internal reference amplification region from the sequencing sample data of methylation-specific amplification sequencing; determining at least one target internal reference amplification region from each internal reference amplification region, and performing standardization processing on the number of amplicons of each target amplification region; calculating the average entropy of each target amplification region according to the proportion of the number of amplicons of each methylation pattern in each target amplification region in each sequencing sample data; inputting the obtained data into a classification model to quickly and accurately obtain the classification result of the sequencing sample data. Improve the accuracy, sensitivity and specificity of early screening classification prediction of cancer.

The invention belongs to the field of aquatic organism breeding, and particularly relates to SNP loci related to grass carp TLR1 genes and aquatic pathogenic bacterium resistance, a locus combination and application of the SNP loci. According to the invention, PCR (Polymerase Chain Reaction) amplification sequencing and PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) technology correlation analysis are carried out to successfully screen out SNP (Single Nucleotide Polymorphism) loci, related to aeromonas hydrophila resistance, of the grass carp TLR1 gene, and the SNP loci are respectively positioned at the 21444058 and the 21444916 basic groups of the 14 # chromosome. Aiming at the two SNP sites, an SNP site combination related to aeromonas hydrophila resistance is further developed, and aeromonas hydrophila resistant individuals of grass carp can be screened by detecting the SNP site combination of the grass carp TLR1 gene. Therefore, the SNP molecular marker provided by the invention can be used for molecular marker-assisted breeding of grass carp, accelerates breeding of disease-resistant grass carp varieties, and provides a reference basis for disease-resistant breeding of grass carp.

The present application relates to the technical field of biotechnology, in particular to a hybridoma cell secreting KOD DNA polymerase monoclonal antibody, the monoclonal antibody and application thereof. The present application utilizes the hybridoma cell to produce the monoclonal antibody capable of specifically binding with KOD DNA polymerase, and the enzyme can be released under the condition of heat activation, so that the polymerization and exonuclease functions are restored. The monoclonal antibody of the present application can be widely applied to PCR amplification, DNA sequencing, gene cloning, protein expression, protein purification and other technologies in the field of molecular biology. The monoclonal antibody can also be used in diagnosis, treatment and prevention and other aspects in the field of biomedicine, such as used for detecting pathogenic microorganisms, tumor markers, gene mutations and the like, and used for gene therapy and protein therapy and the like for treating certain diseases, such as applied to the synthesis of cDNA second strand in the mRNA sequencing library construction process in the second-generation sequencing, so as to improve the sequencing quality and accuracy.

This invention provides a molecular marker associated with sheep weight traits and its application. Primers were designed based on the TNNT1 gene sequence to extract DNA from sheep blood. Through PCR amplification, DNA sequencing, and sequence analysis, a G / C polymorphism site was found at 176 bp of the amplified fragment. Further analysis using AQP primers detected the polymorphism site in 918 Hu sheep and established a least-squares model to analyze the association between genotype and weight traits. Ultimately, the TNNT1 gene fragment amplified by this invention was determined to serve as a molecular marker associated with sheep weight traits. By detecting the genotype of this molecular marker, this invention allows for the selection of GG homozygous sheep for breeding in the core herd, which can improve sheep weight and contribute to increased economic benefits.

The present invention discloses a method for processing samples of lower respiratory tract aspirates of ventilator-associated pneumonia, which belongs to the technical field of processing samples of respiratory aspirates, and solves the technical problem that the processing time of samples of lower respiratory tract aspirates of existing ventilator-associated pneumonia patients is as long as 24-48 hours, and the sample processing efficiency is low. The sample processing method includes: S1, sample processing; including S11. using saponin to remove sample host genes; S12. batch extraction of DNA; S2, construction of multi-sample PCR amplification DNA sequencing library; S3, DNA sequencing in DNA sequencing library; using MinION nanopore sequencer for DNA sequencing, real-time data collection; S4, screening and analyzing the collected data; S5, pathogen gene splicing of processed samples; S6, drug resistance detection of successfully spliced ​​bacteria. The present invention can quickly process samples of lower respiratory tract aspirates of patients with ventilator-associated pneumonia, shortening the sample processing time from the conventional 24 to 48 hours to less than 6 hours.

The application provides PRKAA1 The application provides a gene as a molecular marker related to feed conversion rate of sheep and application thereof, wherein the molecular marker is designed according to PRKAA1 A primer is designed according to the gene sequence, DNA is extracted from sheep blood, and through PCR amplification, DNA sequencing and sequence analysis, a C / T polymorphic site is found at the 165th site of the amplified fragment; further, the polymorphic site of 935 Hu sheep is detected by using KASPar primers, a least square model is established, genotype and feed conversion rate are associated, and finally the application of the amplified PRKAA1 The gene fragment can be used as a molecular marker related to feed conversion rate of sheep. The molecular marker of the application can be used for breeding of high-quality meat sheep with less feed, and provides a genetic engineering means for genetic improvement of feed conversion rate of sheep, and has great practical application value.