23 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.

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 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 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.

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 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.

Disclosed herein include methods, compositions, and kits suitable for use in generation of genome-expanded organisms comprising, e.g., a chimeric genome. In some embodiments, the methods, systems and nucleic acid compositions include systems comprising a donor cell comprising one or more components of a delivery module and a recipient cell comprising one or more components of an integrator module. In some embodiments, the methods, systems and nucleic acid compositions comprise polynucleotides encoding one or more components of a delivery module, and one or more components of an integrator module.

The invention belongs to the field of fish development, and particularly relates to a molecular marker for identifying small yellow croaker gynogenesis filial generation and hybrid filial generation induced by sperms of nibea albiflora as well as a primer group, a kit and application of the molecular marker. The identification primer group of the molecular marker related to the gynogenesis offspring and the hybrid offspring of the small yellow croaker induced by the sperms of the spotted maigre comprises a primer F and a primer 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. The invention further discloses a method for identifying the molecular marker related to the gynogenesis offspring and the hybrid offspring of the small yellow croaker induced by the sperms of the spotted maigre. The molecular marker F / R is developed on the basis of genome data analysis and can effectively identify gynogenesis offspring and hybrid offspring of the small yellow croaker in the juvenile fish stage, and the method comprises the following steps: extracting parent and offspring genome DNA (deoxyribonucleic acid), performing PCR (polymerase chain reaction) amplification and performing agarose gel electrophoresis detection. And finally, distinguishing a gynogenesis individual and a hybrid individual by judging whether the electrophoretic band contains the Nibea albiflora parent marker or not.

The invention provides a primer and a kit for single cell whole genome amplification, and belongs to the technical field of molecular biology. According to the invention, dUTP deoxyuracil nucleotide is used for replacing conventional dTTP for the last basic group of the fixed sequence at the 5'end of the primer, and the primer can be specifically cut off subsequently according to the site, so that the interference of the primer sequence on sequencing is reduced. Meanwhile, compared with commercially available products, the kit comprising the primer is designed, the CV value of the kit is reduced by 63.60%, the kit has good amplification product uniformity and low base preference, the comparison rate in an SE50 sequencing mode is as high as 97.25%, no primer residue interference exists in the amplification process, and the kit is suitable for high-precision single cell sequencing analysis.

The invention discloses a method for determining the stability of polymerase. The invention provides a method for detecting the stability of DNA (deoxyribonucleic acid) polymerase. The stability of the DNA polymerase is detected according to the principle that the DNA polymerase is reversibly inactivated and then activated. According to the scheme, operation is easy and convenient, the requirement for raw materials needed for amplification is low, radioactive isotopes, fluorescent markers and other fluorescent components do not need to be added in the amplification process, and pollution and interference caused by fluorescence are eliminated. The method is more suitable for reaction conditions of reagent development of high-throughput sequencing, whole genome amplification, isothermal amplification, rolling circle replication and the like, is more suitable for judgment of enzyme stability, provides reference for selection of modified enzyme or judgment of use conditions of specific enzyme, is convenient and time-saving, and reduces expenditure.