13 results about "Whole Genome Amplification" patented technology

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.

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 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.

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 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.

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.

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.

The present invention relates to a highly sensitive nested multiplex PCR-based primer panel for the detection of Lumpy Skin Disease Virus (LSDV) in a biological sample obtained from bovine species and livestock animals In particular, the present invention provides a nested multiplex detection primer panel and a kit comprising the same, for the detection of Lumpy Skin Disease Virus (LSDV) and its variants thereof. The present invention also provides a multiplex primer panel for the PCR whole genome amplification and genotyping of Lumpy Skin Disease Virus (LSDV) to obtain genomic data, which can be useful in tracking the evolution of the Lumpy Skin Disease virus and correlating with disease severity. Therefore, the present invention emphasizes its utility in veterinary diagnostics, epidemiological studies, and disease management strategies related to vaccine development for Lumpy Skin Disease Virus (LSDV) infections in livestock.

The present application relates to a method for analyzing the similarity of at least two samples of a plurality of samples comprising genomic DNA. The method comprises the following steps: a) providing a plurality of samples comprising genomic DNA; b) performing a deterministic restriction site whole genome amplification (DRS-WGA) of the genomic DNA separately for each sample; c) preparing a massively parallel sequencing library from each product of DRS-WGA using a no-fragmentation, sequencing adapter / WGA fusion primer PCR reaction; d) performing low-pass whole genome sequencing of the massively parallel sequencing library at an average coverage depth of less than 1x; e) aligning the reads of each sample obtained in step d) to a reference genome; f) extracting the allele content at a plurality of polymorphic loci for each sample; g) calculating a pairwise similarity score locus for at least two samples based on the measured allele content at the plurality of loci; h) determining the similarity of at least two samples based on the similarity score, the method being used for non-invasive prenatal testing or diagnosis.

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.