53 results about "Whole Genome Amplification" patented technology

Methods and systems for sample preparation techniques that prevent inhibition of reactions such as due to the presence of longer oligonucleotides are presented herein. The methods and systems provided herein allow amplification (e.g., whole genome amplification) and sequencing of chromatin accessible regions of single cells.

The invention provides an amplification primer and method for a porcine epidemic diarrhea virus whole genome. The detection primer group for the porcine epidemic diarrhea virus comprises 60 pairs of amplification primers, and the specific sequences of the amplification primers are as shown in SEQ ID NO.1-120. The detection primer group provided by the invention can realize relatively uniform coverage of the porcine epidemic diarrhea virus, and 100% region coverage of a genome is realized in nanopore sequencing. The preparation method of the sequencing fragment of the porcine epidemic diarrhea virus whole genome is simple, convenient and easy to operate and good in amplification effect, nanopore sequencing has the advantages of sequencing and real-time analysis at the same time, the detection time can be greatly shortened, infection of the porcine epidemic diarrhea virus can be rapidly identified and diagnosed, meanwhile, the whole genome sequence of the porcine epidemic diarrhea virus is obtained, and the application prospect is wide. The obtained genome sequence can provide a scientific basis for virus traceability, pathogenic variation tracking, novel strain identification, early warning and the like.

The invention relates to a micro-fluidic chip for sequencing a single virus genome and a sequencing method. The micro-fluidic chip comprises a first functional area, a delay pipeline, a fusion pipeline, a second functional area and a fusion electrode, the first functional area is used for generating first liquid drops of virus-in-oil and lysate; two ends of the delay pipeline are respectively connected with the first functional area and the fusion pipeline; the second functional area is used for generating second liquid drops of the oil bag amplification reaction liquid, and the second functional area is connected with the fusion pipeline to enable the second liquid drops to enter the fusion pipeline; a fusion point is arranged on the downstream of the joint of the fusion pipeline and the second functional area, and the fusion electrode can act on the fusion point to fuse the first liquid drop and the second liquid drop. According to the micro-fluidic chip disclosed by the invention, single-virus whole genome amplification can be completely completed on a single chip, so that the operation steps are simplified, the complexity of sample treatment is reduced, pollution and sample loss are effectively avoided, and the efficiency, the stability and the reliability are improved.

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 invention discloses a method for determining a chromosome copy number and a specific gene carrying chain by using an embryo culture solution. The method can be used for simultaneously detecting embryo chromosome abnormality and a specific gene carrying state. The method comprises the following steps: firstly, collecting blood sample DNA of two specific gene carriers, a pre-certified person or a last generation of relatives; constructing an STR haplotype, designing an STR site primer for amplification, and determining a family haplotype through electrophoretic analysis; the method comprises the following steps: carrying out whole genome amplification on a collected trace free DNA sample of a trace cell or embryo culture solution by using a Pi coPLEX method, respectively designing primers for amplification products aiming at monogenic disease types and mutation sites, and directly detecting pathogenic genes; performing second-generation sequencing, and comparing with a normal sequence to analyze and judge the copy number of chromosomes; primer amplification is designed according to the constructed family distinguishing type STR site, STR site haplotype information is obtained, whether a specific gene variation chain is inherited or not is judged, and therefore an euploid embryo which does not carry a specific gene is efficiently screened out.

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 relates to a primer group for constructing a single cell whole genome sequencing library, the primer group comprises a random primer and an amplification primer, the sequence of the random primer is as shown in SEQ ID No.1, the sequence of the amplification primer is partially the same as that of the random primer, the random primer is used for amplifying whole genome DNA, and the amplification primer is used for amplifying an amplification product of the random primer. The primer group is used for constructing the single-cell whole genome sequencing library, the library construction time is greatly shortened, the library construction process is simplified, and the coverage and uniformity of single-cell whole genome sequencing data are improved.

Disclosed is a method for whole genome amplification and analysis of a plurality of target molecules in a biological sample comprising genomic DNA and target molecules, comprising the steps of: contacting the biological sample with at least one binding agent for at least one target molecule conjugated to a labeled oligonucleotide comprising a binding agent barcode sequence (BAB) and a unique molecular identifier sequence (UMI); performing a separation step to selectively remove unbound binding agent, thereby obtaining a labeled biological sample; simultaneously performing whole genome amplification and amplification of the labeled oligonucleotide on the labeled biological sample; preparing a massively parallel sequencing library from the amplified labeled oligonucleotide; sequencing the massively parallel sequencing library; retrieving the sequences of the BAB and UMI from each sequencing read; calculating the number of different UMIs for each binding agent.

The invention provides a primer pool and a kit for detecting a metapneumovirus whole genome and application of the primer pool and the kit, and belongs to the technical field of biology. The primer pool comprises a first primer pool and a second primer pool, the first primer pool comprises a first primer to a 58 primer, the second primer pool comprises a 59 primer to a 112 primer, and nucleotide series of the first primer to the 112 primer are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 112 in a sequence table. According to the primer pool for detecting the metapneumovirus whole genome, 45 pairs of primers are designed for the metapneumovirus whole genome according to the imbricated principle and are divided into two primer pools, whole genome amplification of metapneumovirus typing can be achieved, 99.65% or above of complete sequences of the metapneumovirus are covered, and the coverage degree is uniform. The method provided by the invention can accurately identify the A or B subtype of metapneumovirus.

The present invention relates to a method for DNA copy- number profiling of a sample comprising genomic DNA First, the genomic DNA is amplified using a deterministic restriction-site whole genome amplification (DRS-WGA). Secondly, an aliquot of said DRS-WGA product is used to prepare a massively-parallel sequencing library with a fragmentation-free, sequencing-adaptor / WGA fusion-primer PCR reaction. Said library is then sequenced to obtain low- pass whole genome sequencing data. The sequencing reads, are finally normalized according to the invention in a way that is cognizant of the DRS-WGA and the sequencing data.

The invention provides a method for carrying out whole genome amplification by taking cell culture fluid as a template, the whole genome amplification is carried out by simulating different cell culture fluids, an optimized whole genome amplification reagent and program are determined, and effective amplification of various culture fluid cells or trace genome templates is realized. Whole genome amplification products are subjected to quality inspection, database establishment and machine loading, and off-machine data are analyzed, so that the effectiveness and the accuracy of whole genome amplification data obtained by the method disclosed by the invention are proved. The problem that an amplification reagent on the current market cannot resist inhibition amplification is solved.

The invention relates to a method and a system for quality control of single cell whole genome amplification products. The method comprises detection of different chromosome segments. The invention also relates to reagents used in the method.

Disclosed is a method for whole genome amplification and analysis of multiple target molecules in a biological sample containing genomic DNA and target molecules, the method comprising the steps of: contacting the biological sample with at least one binder for at least one of the target molecules, the binder being conjugated to a tagged oligonucleotide comprising a binder barcode sequence (BAB) and a unique molecular identifier sequence (UMI); obtaining a labeled biological sample by performing a separation step to selectively remove unbound binders; simultaneously performing whole genome amplification and amplification of the tagged oligonucleotides on the labeled biological sample; creating a massively parallel sequencing library from the amplified tagged oligonucleotides; sequencing the massively parallel sequencing library; searching for the sequences of the BAB and UMI from each sequencing read; and counting the number of different UMIs for each binder.

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.

The application belongs to the field of single cell sequencing, and discloses a method for constructing a microbial single cell genomics sequencing library, which comprises the following steps: 1) lysing microbial single cells; 2) adding an amplification reaction mixture to perform whole genome amplification; 3) adding a fragmentation reagent to obtain a fragmented amplification product; and 4) adding microbeads and an amplification reaction mixture to label the fragmented reaction product with a barcode, so as to obtain raw materials for constructing a microbial single cell whole genome sequencing library. The effective library molecules obtained by using the transposase complex linker sequence and the library construction method designed in the application can be compatible with an Illumina second-generation sequencer, and can be mixed with other sample libraries for sequencing, so that the sequencing cost is greatly reduced.

The present invention belongs to the field of gene detection, and specifically relates to a primer set for whole genome amplification of monkeypox virus and its application. The present invention provides a primer set for whole genome amplification of monkeypox virus, the primer set comprising: a first primer set, which comprises at least two selected from the sequences shown in SEQ ID NO:1-178; a second primer set, which comprises at least two selected from the sequences shown in SEQ ID NO:179-358. The primer set provided by the present invention for monkeypox virus has better sensitivity, can achieve whole genome amplification of monkeypox virus, the proportion of fragments with gene coverage of more than 30× is more than 99.9%, and the sequencing depth can reach up to 90000×. The present invention has broad application prospects in monitoring monkeypox virus subtypes.

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 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.

Methods and systems for sample preparation techniques that prevent inhibition of reactions such as due to the presence of longer oligonucleotides are presented herein. The methods and systems provided herein allow amplification (e.g., whole genome amplification) and sequencing of chromatin accessible regions of single cells.

The invention relates to the field of nucleic acid amplification, in particular to a method for isothermal amplification of nucleic acid molecules. The invention provides a method for isothermal amplification of nucleic acid molecules. The method comprises the following steps: mixing nucleic acid to be amplified with helicase to obtain single-stranded nucleic acid; mixing the single-stranded nucleic acid with the single-stranded binding protein and the primer enzyme to obtain oligonucleotide; under the condition that dNTP exists, oligonucleotide and DNA polymerase are mixed, and an extension product is obtained; repeating the steps for one or more times on the extension product to obtain the sequence of the nucleic acid to be amplified. The HPA disclosed by the invention is an isothermal amplification method which comprises the following steps of: unfastening double strands of a template by utilizing helicase, generating a primer by utilizing primer enzyme, and then amplifying by utilizing DNA (Deoxyribose Nucleic Acid) polymerase. According to the method, no additional primer needs to be added, non-specific amplification products such as primer dimers are prevented from being generated, high-temperature denaturation is also not needed, and the reaction can be completed at a constant temperature. Whole genome amplification can be carried out on an original template, and the DNA concentration of the original template is improved, so that downstream detection is facilitated.

Disclosed are methods of monitoring minimum residual disease using genomic profiling. In some methods, a subject has or has had multiple myeloma or a precursor thereof. The methods include amplifying a sample using primary template-directed whole genome amplification (PTWGA) and enable monitoring of liquid samples, such as blood samples.

The present invention provides an isolated peptide of SEQ ID NO: 1 needed for primase active as well as new replicative DNA polymerase enzymes, preferably that of SEQ ID NO: 2, comprising said peptide. Thus, these DNA polymerases are endowed with priming activity and do not require externally provided primers for initiating and performing DNA amplification. These polymerases are able to carry out a faithful and processive de novo DNA synthesis of DNA templates in the absence of pre-synthetized primers. Therefore, these enzymes of the invention act both as primases and DNA polymerases. Furthermore, they show translesion synthesis capacity, so that they may be useful not only for whole-genome amplification but also for the amplification of damaged DNAs. The invention further refers to methods for amplifying templates DNAs using these enzymes.

A method for analyzing loss of heterozygosity (LoH) in at least one sample containing genomic DNA is disclosed, the method comprising the following steps: a. providing at least one sample containing genomic DNA; b. performing deterministic restriction site whole genome amplification (DRS-WGA) of the genomic DNA; c. preparing a massively parallel sequencing library from the product of the DRS-WGA; d. performing low-pass whole genome sequencing on the massively parallel sequencing library with an average coverage depth of <1; e. aligning the reads obtained in step d on the reference genome of the at least one sample; f. extracting the allele content of multiple loci, wherein the multiple loci include polymorphic loci and / or heterozygous loci; g. assigning a LoH score to at least one genomic window of the reference genome for the at least one sample based on the number of loci having at least two different alleles among the multiple loci.

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 provides a single cell transposase library building method, and belongs to the technical field of biology. According to the method provided by the invention, the specific primer pre-amplification step is added after the transposase is broken, so that the copy number of a single cell nucleic acid sample can be amplified, the problems of low coverage and the like when a traditional transposase library building method is applied to single cells are solved, whole genome amplification is not needed, the operation time is effectively shortened, and the operation efficiency is improved. And the obtained sequencing data is higher in uniformity.

Methods and systems for array-based methods for genotyping multiallelic markers are disclosed. Also disclosed herein are methods for whole genome amplification and locus specific multiplex PCR for selectively biasing amplification for reducing effects of undesired pseudogenes in resulting data.

This genetic analysis method comprises: isolating fixed maternal blood cells one by one; de-crosslinking DNA from protein in a proteinase-containing buffer for each of the isolated blood cells; extracting the de-crosslinked DNA; subjecting the extracted DNA to whole genome amplification; and amplifying a target sequence by using a plurality of specific markers from the amplified whole genome amplification product.

The application relates to the field of forensic identification, and discloses a microhaplotype genetic marker system for mixed stain inspection and a method and application thereof. The system contains 135 microhaplotype sites of autosomes and corresponding amplification primers, and a microhaplotype typing system based on MPS technology is constructed, which has good sensitivity, specificity and inhibitor tolerance. Further combined with a microfluidic chip, automatic capture and whole genome amplification of single cells are realized, and the average locus detection rate of single cell samples reaches 83.48% in cooperation with a short fragment microhaplotype marker system, and individual sources in complex mixed stains can be accurately distinguished. The system can be widely applied to scenes such as individual identification, kinship identification and mixed stain analysis in forensic science.

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.