35 results about "Genome amplification" patented technology

The invention discloses a method for identifying Yushan black pigs by adopting an SNP (Single Nucleotide Polymorphism) molecular marker technology, and particularly relates to the technical field of molecular detection.The method comprises the following steps: S1, sample preparation: collecting any one of ear tissues, tail tips or blood samples of to-be-detected pig individuals, and extracting to obtain genome DNA (Deoxyribose Nucleic Acid); the genome DNA is used as a template, amplification detection is conducted through a first primer pair, a second primer pair, a third primer pair and a fourth primer pair corresponding to the first SNP site, the second SNP site and the fourth SNP site, and the nucleotide sequences of the first primer pair, the second primer pair and the fourth primer pair are sequentially SEQ ID NO.5-SEQ ID NO.12. By detecting the specific single nucleotide polymorphic sites in the Yushan black pig genome and carrying out genetic typing comparison, the Yushan black pigs and the filial generation thereof can be quickly and accurately identified.

The application belongs to the field of fish development, and particularly relates to a molecular marker for identifying Nibea albiflora sperm-induced Pseudosciaena heteroclita gynogenesis offspring and hybrid offspring, a primer set thereof, a kit and application. The primer set for identifying the molecular marker of the Nibea albiflora sperm-induced Pseudosciaena heteroclita gynogenesis offspring and hybrid offspring comprises primers F and R, the nucleotide sequence of the primer F is shown as SEQ ID NO. 1, and the nucleotide sequence of the primer R is shown as SEQ ID NO. 2. Based on genomic data analysis, the application develops the molecular marker F / R which can effectively identify the Pseudosciaena heteroclita gynogenesis offspring and hybrid offspring in the juvenile stage. The method comprises extracting genomic DNA of parents and offspring, PCR amplification and agarose gel electrophoresis detection, and finally distinguishing the gynogenesis individuals and hybrid individuals by whether the Nibea albiflora parent marker is contained in the electrophoresis band.

The application discloses a set of general primers for amplifying the whole genome of the mitochondrion of Trionychidae, and belongs to the technical field of molecular biology; the specific steps comprise the following steps: extracting the total DNA of the genome of Trionychidae, designing the general amplification primers of the whole sequence of the mitochondrial genome, and performing PCR amplification and sequencing on the mitochondrial genome DNA. The amplification primers for the whole genome sequence of the mitochondrion of Trionychidae are composed of 11 pairs of single-stranded oligonucleotides. The general amplification primers of the application can be used to quickly and efficiently amplify the whole sequence of the mitochondrial genome of various Trionychidae, and provide a favorable technical means for the identification of unknown germplasm of Trionychidae, the protection of species resources, and the research on the system evolution.

The invention relates to a method of determining somatic allele-specific copy number alterations (CNAs) in the genomes of cells in a test-sample from a subject, the method comprising: i) providing an indexed-DNA library of DNA fragments resulting from whole-genome amplification of genomic DNA from cells of the test-sample, ii) providing whole genome sequencing data of reference non-cancer cells from a reference-sample from the subject; and iii) determining somatic allele-specific copy number alterations in the genome(s) of the cells of the test-sample; and associated methods and uses in cancer therapy.

The application discloses a single cell whole genome amplification sequencing method. The application comprises the following steps: 1) separating a single cell, adding a single cell lysis solution to sufficiently lyse the single cell and digest proteins combined on the genomic DNA; 2) using a Tn5 transposome with a specific adapter sequence to fragment the DNA, while adding adapters to both ends of the fragments; 3) after end repair, using a ribonuclease to cut modified ribonucleotide residues on the adapter sequence; 4) using the adapter sequence as a primer to linearly amplify the fragmented DNA by using a single primer PCR method; and 5) adding sequencing adapters to both ends of the DNA fragments by using a chain extension and PCR method to obtain a final sequencing library. The method disclosed by the application can amplify single cell whole genome DNA with high coverage, uniformity and fidelity, and simultaneously and accurately detect chromosomal copy number variation CNV and single nucleotide mutation SNV on a single cell genome.

The present application relates to a primer combination for amplifying whole genome DNA of sheep embryo cells and application thereof, and the primer combination is composed of 20 or 30 primers. The present application designs random primers based on sheep reference genome sequence, optimizes the reaction system based on MDA whole genome amplification technology, so that the whole genome DNA amplification product with high coverage and low mismatch rate can be generated by using 10 sheep embryo cells or as low as pg level genomic DNA, which can be directly adapted to downstream applications such as second-generation sequencing and targeted capture sequencing, and the detection cost is reduced.

The application discloses a single-cell mitochondrial whole-genome efficient and specific amplification method. The application provides a single-cell or multi-cell mitochondrial genome DNA high-fidelity amplification method, which comprises the following steps: taking total DNA obtained by lysing a test single cell as a template, using a chimeric primer composed of a 5' end non-template matching common sequence and a 3' end mitochondrial DNA (mtDNA) specific matching sequence, and a primer corresponding to the 5' end non-template matching common sequence of the chimeric primer, and using a high-fidelity DNA polymerase with strand displacement activity to simultaneously amplify several overlapping amplicons which completely cover the single-cell or multi-cell mitochondrial genome DNA. The application avoids various problems in the current single-cell mtDNA in-vitro amplification approach, and is a very reliable mtDNA whole-genome amplification method.

The invention develops a method for absolute quantification of fungal population based on a Pacbio three-generation long fragment sequencing platform and application thereof, and belongs to the technical field of microbial amplicon detection. According to the method, the synthesized chimeric DNA is used as an internal standard, and a fungal community in a sample is absolutely quantified on the basis of an amplicon high-throughput sequencing platform. The specific method comprises the following steps: directly adding the synthesized chimeric DNA into an environmental sample; extracting genome DNA of the environment sample; carrying out high-throughput sequencing on the ITS full-length sequence of the fungus through PCR amplification; the high-throughput sequencing results calculate the relative abundance and absolute abundance of the amplicon family (e.g., the copy number of fungal ITS per unit mass sample). A highly complex environmental sample is taken as a research object to prove that the method can sensitively and accurately quantify the absolute abundance of a specific group. The method is widely applicable to microbial community analysis of samples from different sources, including environmental samples from soil, samples from plant tissues, samples from human intestinal tracts, and samples from food samples. The method disclosed by the invention is simple to operate, accurate in detection and wide in application, and has a wide market prospect in identification and analysis of microbial communities.

The invention relates to a mantle cell lymphoma BCL1-IGH rearrangement MRD detection system. The detection system comprises: (1) a sample module, which is used for extracting peripheral blood or marrow genome DNA of a sample to be detected; (2) an amplification sequencing module: performing amplification by taking the genome DNA as a template to obtain an amplification product, and performing high-throughput sequencing on the amplification product; and (3) the bioinformatics analysis module is used for carrying out BCL1-IGH rearrangement cloning identification and cloning tracking on the to-be-detected sample according to the sequencing result. According to the detection system provided by the invention, the detection process is remarkably simplified, the detection cost and the analysis difficulty are reduced, the detection rate of minimal residual focuses of mantle cell lymphoma is high, and the detection system can be applied to the fields of auxiliary clinical diagnosis and the like.

This invention discloses a kit for amplifying the whole genome of bovine embryonic cells and its applications. The invention provides a primer set for amplifying the whole genome of bovine embryonic cells, consisting of 20 primers, each 6 nt in length. This invention can utilize 4-8 bovine embryonic cells or genomic DNA down to the pg level to amplify and generate whole-genome DNA amplification products with high coverage and low mismatch rate. These products can be directly adapted to downstream applications such as next-generation sequencing and targeted capture sequencing, while reducing detection costs. Furthermore, the three-step operation process of this invention (cell lysis-lysis termination-isothermal amplification) can complete efficient amplification within 3.5 hours.

This invention provides a primer set, library construction method, and application for whole-genome detection of porcine reproductive and respiratory syndrome virus (PRRSV). The primer set comprises 16 primer pairs, and the sequences of the upstream and downstream primers for each primer pair are shown in SEQ ID NO. 1 to SEQ ID NO. 32. The primer set of this invention exhibits good specificity and sensitivity, and shows promising application prospects in the amplification of the entire PRSV genome and the preparation of reagents or kits required for amplification. The library construction method of this invention includes multiplex PCR amplification using the primer set described herein. This method is compatible with next-generation sequencing platforms and nanopore sequencing platforms, offering a high degree of automation. Even in samples with viral concentrations as low as 1 copy / μL, it can obtain more than 95% of the PRSV sequence, with a sequencing depth exceeding 1000X.

The invention discloses a pigeon mitochondrial whole genome amplification and assembly method. The method comprises the following steps: (1) extracting genome DNA of a to-be-detected pigeon; (2) carrying out PCR (Polymerase Chain Reaction) amplification through the primers to obtain two sections of pigeon mitochondrial DNA (Deoxyribose Nucleic Acid) fragments, and (3) carrying out high-throughput sequencing; and (4) assembling the mitochondrial genome to obtain the mitochondrial DNA full length.

The present invention provides universal primers and a method for amplifying the whole mitochondrial genome of mites from the family Acaridae. This invention, for the first time, provides a set of universal primers for amplifying the whole mitochondrial genome of mites from the family Acaridae. By dividing the mitochondrial genome into six overlapping fragments, conventional PCR amplification, Sanger sequencing, and sequence splicing are performed to rapidly obtain the complete mitochondrial genome sequence of mites from the family Acaridae. This method requires relatively low instrumentation and analytical equipment, making it easy to implement. Furthermore, the entire mitochondrial genome sequence can be obtained from the total DNA of a single individual, avoiding interference from the genomic DNA of multiple individuals. This method has been validated in the amplification of the mitochondrial genomes of six species of mites from the family Acaridae. This method is of great significance for obtaining more complete mitochondrial sequences of mites from the family Acaridae, further inferring the phylogenetic relationships between the genera of the family Acaridae, and the phylogenetic relationships between the families of the family Acaridae. It also provides a reference for studying the evolution and phylogenetic relationships of the Acari.

The invention belongs to the technical field of biology, and particularly relates to a DNA polymerase mutant for improving long fragment amplification efficiency and application of the DNA polymerase mutant. According to the invention, the DNA polymerase mutants KODL and KODW with enhanced continuous synthesis capability are successfully constructed by carrying out combined mutation of specific amino acid sites on the KOD DNA polymerase, the amplification efficiency of the mutants on a long-fragment DNA template (such as 30kb) is remarkably improved on the basis of retaining the amplification efficiency on fragments with conventional lengths, and the application of the mutants to the preparation of the long-fragment DNA template is facilitated. The technical bottleneck of the existing variable temperature amplification enzyme in long fragment amplification is broken through, and an efficient and reliable enzymology tool is provided for molecular biology application of long fragment gene cloning, whole genome amplification, complex structure analysis and the like.

The present invention relates to a genome amplification module having a branch space adjacent to an inlet through which an extract flows, and provides a genome amplification module in which a branch space that is wider and deeper than the extract flow passage is formed adjacent to the inlet, and after the branch space is filled with extract, the extract can be simultaneously injected along each extract flow passage.

The application belongs to the technical field of biology, and discloses a primer group, a kit and a method for quality control of whole genome amplification of Macaca fascicularis. The quality control method for the whole genome amplification product of Macaca fascicularis is simple and easy to operate, 6 pairs of primers can be used in the same reaction tube, thereby reducing the steps of adding reagent components and sub-packaging, and the whole reaction has low requirements on laboratory conditions, only a conventional PCR amplification laboratory, agarose electrophoresis and a gel analyzer are needed.

The present application relates to the assisted evaluation method of the second biopsy in the pre-implantation genetic testing. The blastocyst first biopsy process collects and cryopreserves the corresponding biopsy droplet; for the blastocyst with CNV detection failure or suspected abnormal CNV detection result in the first biopsy cell, the first biopsy droplet is thawed to complete genome amplification, CNV library construction, high-throughput sequencing and CNV analysis, and the second biopsy suggestion is given according to the detection result. If the biopsy droplet detects the abnormal aneuploidy or chimera clinically interpreted, it is suggested that the corresponding blastocyst has a high probability of chromosomal abnormality, and the second biopsy is not recommended; if the biopsy droplet does not detect the abnormal aneuploidy or chimera clinically interpreted, it is suggested that the corresponding blastocyst has a high probability of chromosomal normality, and the second biopsy is recommended. The present application proves that the first biopsy droplet contains detectable genetic material, and the consistency of the chromosomal ploidy detection result and the biopsy cell is more than 80%, which can assist the second biopsy decision and improve the decision efficiency and scientificity of PGT detection.

The invention relates to a kit for detecting nocardia seriolae of jewfish based on RPA-CRISPR / Cas12a as well as a detection method and application of the kit. The main component of the kit is a composition for identifying nocardia seriolae, and the composition comprises a specific primer for detecting nocardia seriolae based on RPA-CRISPR / Cas12a, a Cas12a protein, crRNA and a fluorescence reporter probe ssDNA Reporter, wherein the specific primer, the Cas12a protein, the crRNA and the fluorescence reporter probe are used for detecting nocardia seriolae based on RPA-CRISPR / Cas12a. The detection method of the kit comprises the following steps: S1, extracting a sample genome DNA; s2, carrying out RPA (recombinase polymerase amplification); s3, CRISPR (clustered regularly interspaced short palindromic repeats) / And S4, interpreting a detection result. According to the invention, a target gene segment of nocardia seriolae is rapidly amplified at a constant temperature by virtue of an RPA technology, and the target gene segment of nocardia seriolae is specifically targeted by virtue of accurate recognition and trans-cleavage capabilities of CRISPR / Cas12a, so that rapid detection visible to naked eyes is realized. Compared with conventional detection methods such as PCR, qPCR and the like, the kit does not need a professional thermal cycler, has the advantages of high sensitivity, strong specificity, simplicity and convenience in operation, low cost and the like, and is expected to be applied to on-site instant detection of aquaculture sites and basic laboratories.

The invention provides a kit for detecting jewfish aeromonas veronii based on RPA-CRISPR / Cas12a, a detection method and application of the kit. The main component of the kit is a composition for identifying the aeromonas veronii, and the composition comprises a specific primer for detecting the gyrB gene of the aeromonas veronii based on RPA-CRISPR / Cas12a, a Cas12a protein, crRNA and a fluorescence report probe ssDNA Reporter. The detection method of the kit comprises the following steps: S1, extracting a sample genome DNA; s2, carrying out RPA (recombinase polymerase amplification); s3, CRISPR (clustered regularly interspaced short palindromic repeats) / And S4, interpreting a detection result. According to the invention, a target gene segment of the aeromonas veronii is rapidly amplified at a constant temperature by virtue of an RPA technology, and the target gene segment of the aeromonas veronii is specifically targeted by virtue of accurate recognition and trans-cleavage capabilities of CRISPR / Cas12a, so that rapid detection visible to naked eyes is realized. Compared with conventional detection methods such as PCR, qPCR and the like, the kit does not need a professional thermal cycler, has the advantages of high sensitivity, strong specificity, simplicity and convenience in operation, low cost and the like, and is expected to be applied to on-site instant detection of aquaculture sites and basic laboratories.

The invention develops a bacterial population absolute quantification method based on a Pacbio three-generation long fragment sequencing platform and application thereof, and belongs to the technical field of microbial amplicon detection. The synthetic chimeric DNA is used as an internal standard, and the microbial community in the sample is absolutely quantified at the same time. The specific method comprises the following steps: directly adding the synthesized chimeric DNA into an environmental sample; extracting genome DNA of the environment sample; carrying out PCR amplification on the 16S full-length sequence and carrying out high-throughput sequencing; and calculating relative abundance and absolute abundance of the amplicon family according to a high-throughput sequencing result. A highly complex environmental sample is taken as a research object to prove that the method can sensitively and accurately quantify the absolute abundance of a specific group. The method is widely applicable to microbial community analysis of samples from different sources, including environmental samples from soil, samples from plant tissues, samples from human intestinal tracts, and samples from food samples. The method disclosed by the invention is simple to operate, accurate in detection and wide in application, and has a wide market prospect in identification and analysis of microbial communities.

The application provides a kind of based on four base microsatellite fluorescence multiplex PCR red claw crayfish parentage identification method, belong to biological engineering technical field, the application includes the following steps: (1) extracting the genome DNA of sample to be measured;(2) the screening of red claw crayfish microsatellite primer (3) the construction of multiplex PCR amplification system 20 pairs of primers in step (2) are grouped according to 120-200bp, 220-260bp, 280-340bp, 360-420bp, 440bp-500bp interval, select amplification combination, obtain multiple multiplex PCR amplification system;(4) parentage identification sample genome DNA amplification, obtain multiplex PCR product, typing, read parent and offspring genotype, judge parent-child relationship.The application can realize that there is no interaction between multiple primers, no non-specific amplification, do not make the amplified product overlap.In the process of PCR amplification, multiple target fragments are amplified simultaneously, which can save experimental samples, experimental cost, improve experimental efficiency, and achieve 100% parentage identification success rate for red claw crayfish.

According to the microfluidic single RNA virus genome amplification method based on the RT-BST system, an RT-BST amplification system is introduced into a microfluidic liquid drop system, reverse transcription and strand displacement amplification of a single RNA virus in liquid drops are achieved for the first time through Bst DNA polymerase, and the technical problems that an RNA template is unstable, the amplification efficiency is low and the coverage rate is insufficient are solved. According to the RT-BST technology, the continuous reaction from RNA to cDNA and then to whole genome amplification is completed in a single droplet, the experimental process is greatly simplified, pollution and loss caused by multi-step operation or sample transfer are avoided, and the detection sensitivity and the result reliability are remarkably improved. Compared with a traditional MDA amplification technology mainly suitable for DNA viruses, the RT-BST method disclosed by the invention shows excellent applicability to RNA viruses and shows broad-spectrum compatibility.

A mutant Taq DNA polymerase and its application belong to the field of biotechnology. On the one hand, the present invention provides a mutant Taq DNA polymerase. Compared with the amino acid sequence of the wild-type Taq DNA polymerase, the mutant Taq DNA polymerase includes the mutation site D785 and / or the mutation site R573. On the other hand, the present invention provides the application of the mutant Taq DNA polymerase in the preparation of PCR reaction reagents. The Taq DNA polymerase mutant described in the present invention is a mutant obtained by predicting the activity of key mutation sites by AlphaFold3 and then verifying the expression. The double mutant strain obtained in the present invention provides a better Taq enzyme selection for genomic amplification.

The invention discloses a method for constructing a linkage analysis library based on single sperms or polar bodies. The method comprises the following steps: splitting a single sperm or a polar body to prepare a split single sperm or polar body; and carrying out multiple PCR amplification, terminal repair, linker connection and PCR enrichment and purification to prepare the target library. The method at least has the advantages that single cell whole genome amplification does not need to be carried out, library construction can be directly carried out on a polar body or a single sperm, the detection period is shortened, and the detection cost is reduced.

The invention belongs to the technical field of gene detection, and discloses a primer group for human mitochondrial whole genome amplification, a kit, a library construction method and application, the primer group comprises 128 pairs of primers, the nucleotide sequences of forward primers in the 128 pairs of primers are sequentially shown as SEQ ID NO.1-SEQ ID NO.128, and the nucleotide sequences of reverse primers in the 128 pairs of primers are sequentially shown as SEQ ID NO.129-SEQ ID NO.256. The kit comprises the primer group, an amplification buffer solution, two enhancers, a universal primer and a specific primer. The library construction method comprises the following steps: acquiring DNA of a sample to be detected, performing two rounds of PCR amplification to obtain a mitochondrial genome library, and performing sequencing to obtain sequencing data. According to the method, more than 99% of the full length of human mitochondria can be covered, higher sequencing depth can be obtained under the condition of extremely small data volume, and the sequencing cost is greatly reduced.

The invention provides a single-cell genome amplification library building method, which comprises the following steps of: splitting a single cell; dNA fragmentation: a cleavage product is subjected to DNA fragmentation with at least two restriction endonucleases, most recognition sites of the first restriction endonucleases are located in a high-CG region of the genome, and recognition sites of the second restriction endonucleases do not have CG; sequentially carrying out terminal repair A addition, linker addition and User enzyme treatment on the fragmented product; and library amplification: adding a sequence tag into the User enzyme treatment product, and carrying out DNA amplification, thereby completing single cell genome amplification and library construction in one step. The invention also provides a single cell genome amplification library building kit, which comprises a fragmentation enzyme combination, and the fragmentation enzyme combination comprises a second restriction enzyme without CG in first restriction enzyme recognition sites of which most recognition sites are located in a genome high CG region. According to the method and the kit, the sequencing success rate after library establishment can be greatly improved.

This application belongs to the field of nucleic acid detection technology, and particularly relates to a method and kit for co-detecting bovine mastitis resistance and early embryo gender. This application uses a self-made lysis solution and neutralization solution to pretreat the sample, performs whole-genome amplification using Phi29 enzyme, then conducts multiplex PCR amplification based on the developed primer system, and finally combines capillary electrophoresis to achieve co-detection. This application only requires 4-10 embryonic cells to meet the detection requirements, realizing the synchronous detection of bovine early embryo gender and bovine mastitis resistance, and having advantages such as high sensitivity and strong specificity.

The present invention relates to a genome amplification module having a branch space adjacent to an inlet through which an extract is introduced, the genome amplification module in which a branch space is formed in a portion adjacent to an inlet and is wider and deeper than an extract moving passage so that the extract can be simultaneously injected along each extract moving passage after the extract is filled in the branch space.

The present invention provides a primer set, a library construction method and an application for detecting porcine epidemic diarrhea virus based on whole genome. The primer set includes 27 pairs of primers, and the sequences of the upstream primers and downstream primers of the 27 pairs of primers are shown as SEQ ID NO. 1 to SEQ ID NO. 54. The primer set of the present invention has good specificity, sensitivity and repeatability, and has good application prospects in the amplification of the whole genome of porcine epidemic diarrhea virus and the preparation of reagents or reagent kits required for amplification. The library construction method of the present invention includes performing multiplex PCR amplification using the primer set described in the present invention; the library construction method of the present invention is adapted to second-generation sequencing platforms and nanopore sequencing platforms, with high automation. Even in samples with a virus content as low as 1 copies / μL, more than 95% of the porcine epidemic diarrhea virus sequences can be obtained, and the sequencing depth can reach more than 1000X.

The present invention relates to a genome amplification module having a branch space adjacent to an inlet through which an extract is introduced, in which a branch space is formed in a portion adjacent to the inlet, the branch space being wider and deeper than an extract movement path, the branch spaces are arranged such that after the extract is filled into the branch spaces, the extract can be injected simultaneously along each extract movement path.