46 results about "DNA Contamination" patented technology

Problems to be Solved: The present invention provides a simpler and less expensive method for purifying physiologically active proteins, especially antibodies, which can ensure removal of impurities such as DNA contaminants and viruses, and which can minimize a loss of physiologically active proteins. Means for Solving the Problems: A method for removing impurities in a physiologically active protein-containing sample, which comprises the following steps: 1) allowing the physiologically active protein-containing sample to be converted into an aqueous solution of low conductivity at a pH below the isoelectric point of the physiologically active protein; and 2) removing the resulting particles.

The invention belongs to the field of transcriptome analysis and relates to a quick single-cell RNA (ribonucleic acid) reverse transcription and library construction method. According to the method, 20-500ng high-quality full-length double-chain cDNA (complementary deoxyribonucleic acid) is amplified by taking 1-2000 cells or 10pg-20ng extracted eukaryote total RNA as an initial, and a high-quality cDNA library meeting downstream analysis requirements is obtained. The method effectively avoids 3' preference and genome DNA contamination in a cDNA synthesis process; an expression quantitation molecule label can assist in gene expression quantity calculation; and at the same time, the expression quantitation molecule label can also keep chain source information during complete amplification of RNA sequence information. The method can achieve a reverse transcription and amplification library construction success rate of above 95%; the cDNA library can be connected with an Illumina main stream sequencing platform; lower computer data (5M Reads) can detect gene expression of above 90%; the gene expression consistency exceeds 90%; amplification has no obvious bias; and a required sample input amount is smaller.

The invention discloses a method for detecting a variable spliceosome in a third generation full-length transcriptome. The method comprises the following steps: merging original annular test sequences with joints removed to form a monomolecular transcript sequence, and screening a third generation full-length transcript sequence; comparing the third generation full-length transcript sequence with a reference genome sequence, and screening a third generation full-length transcript sequence having coverage and similarity with the reference genome sequence larger than preset thresholds; carrying out splicing false positive filtration and DNA contamination filtration on the screened third generation full-length transcript sequence; and carrying out gene annotation and variable spliceosome annotation on the filtered third generation full-length transcript sequence. An overlong read length of a third generation sequencing technology mentioned in the method disclosed by the invention is large enough to cover most RNA, the third generation full-length transcript sequence can be obtained by SMRT sequencing transcriptomes without being assembled, and a splicing structure of a gene can be effectively obtained by third generation transcriptome sequencing, and more perfect gene model annotation can be constructed.

The present invention provides a novel robust, sensitive, reproducible, and accurate method of detecting and quantifying host cell genomic DNA contamination utilizing quantitative real time Polymerase Chain Reaction (qPCR), wherein the qPCR primers are complementary to the highly repetitive host cell genomic DNA sequences, e.g., Alu-equivalent sequences. The present invention is particularly useful for determining the levels of residual genomic DNA in biological products to be administered as therapeutics, e.g., therapeutic proteins.

The present invention relates to a simple, effective and quick RNA extraction method for absorbing RNA by utilizing a silicon film column. The method includes the following steps: after cells are collected, denaturing solution and chloroform are added into the cells, uniformly mixed and centrifugated until the bacteria solution is divided into three layers in a tube; the top layer of liquid is extracted and transferred into the silicon film column to be centrifugated; the previous step is repeated; centrifugation is conducted again in order to ensure that no liquid exists in the silicon film column; double distilled water and eluent are added into the silicon film column; and after centrifugation, the eluent is collected into a clean centrifugal tube. The denaturing solution contains split agent, RNA extraction agent and water; wherein, the split agent is guanidinium isothiocyanate, sodium dodecyl sarcosinate or phenol; and the RNA extraction agent is sodium citrate, sodium acetate, sodium lactate, ascorbic acid, potassium malate, sodium oxalate or potassium tartrate. The extraction time of the method is not longer than 6 minutes, DNA pollution cannot be generated, the extraction process can be easily automated, and the purity of the extracted RNA is high.

The invention belongs to the technical field of analysis of gene expression and particularly relates to application of ClCAC gene and ClSAND gene as reference genes in analysis of gene expression of watermelon fruits. According to the invention, specific primers are designed for ClCAC gene and ClSAND gene. The specific primers cross over joint loci of two exons; no amplification product is generated when a genome DNA is used as a template; a target segment can be obtained through amplification when cDNA is used as a template; the nucleotide sequences of the primers are shown in SEQ ID No.1-4. Real-time fluorescence quantification PCR detection shows that both the ClCAC gene and the ClSAND gene can be expressed stably in different development stages of watermelon fruits which are different in gene type and fruit bearing mode and thus the ClCAC gene and the ClSAND gene can be used as a reliable reference gene combination in analysis of gene expression by real-time fluorescence quantification PCR detection. The reference gene combination has the advantages of good expression stability and the like and is not influenced no matter whether the genome DNA contamination exists in a cDNA sample or not.

The present invention pertains to a method of isolating RNA from a sample comprising RNA, and DNA, comprising: a) adding an acidic denaturing composition comprising a chaotropic agent and phenol to the sample; b) adding a water-insoluble organic solvent and separating the resulting phases thereby forming a multi-phase mixture comprising an aqueous phase, optionally an interphase and an organic phase, wherein the RNA is concentrated in said aqueous phase and DNA and proteins are concentrated in said organic phase and/or in said interphase; and c) isolating said RNA from said aqueous phase, wherein at least one cationic detergent is added before separating the phases. It was found that the addition of at least one cationic detergent considerably reduces the amount of DNA in the aqueous, RNA containing phase. Therefore, the present invention allows to easily isolate pure RNA which comprises considerably less DNA contaminations.

The invention provides a bacteriophage genome DNA (deoxyribonucleic acid) extraction kit and method. The kit comprises a reagent A (chloroform), a reagent B (DNase I), a reagent C (RNase A), a reagent D (precipitation buffer solution), a reagent E (pyrolysis buffer solution), a reagent F (primary pyrolysis main solution), a reagent G (secondary pyrolysis solution), a reagent H (impurity removal solution), a reagent I (DNA combination buffer solution), a reagent J (rinsing buffer solution) and a reagent K (DNA eluate). The extraction method comprises the following steps: removal of bacterium cells and fragments, degradation of bacterium nucleic acid, bacteriophage particle precipitation, bacteriophage capsid protein structure damage and hydrolysis, impurity removal, DNA liquid separation, DNA adsorption, cleaning of adsorbed DNA and elution of adsorbed DNA. The kit and method can be widely used for extracting the bacteriophage genome DNA, and has the advantages of high extraction speed, high extracted DNA yield and high purity, and the extracted DNA can not be polluted by the host bacterium genome DNA.

The present invention relates to method of extracting ancient human remains matter DNA. The method includes first treating skeleton sample with plasma to decompose the surface pollutant and subsequent DNA extraction. The DNA extraction includes the steps of polishing skeleton sample, hypochloric acid treatment, ultraviolet irradiation, alcohol solution washing to eliminate foreign microbe contamination, Ar/O2 (9:1) plasma treatment, grinding in liquid nitrogen to obtain powder sample; digesting and cracking sample, phenol-chloroform extraction to eliminate digested organic molecule, adsorption with silica adsorption column, and filtering in 3 KDa ultrafiltering centrifugation tube to obtain ancient human remains matter DNA. The method of the present invention can eliminate surface pollutant fast effectively, and has high ancient human remains matter DNA extracting success rate and high template content.

The invention relates to the technical field of blood sample preservation, and particularly discloses a free DNA blood preservative for preventing hemolysis and applications thereof. Each 100ml of theblood preservative comprises the following ingredients: 40.0-100.0g of imidazolidinyl urea, 4.0-20.0g of EDTA-3K, and 4.0-10.0g of glycine. The blood preservative of the invention can reduce the hemolysis phenomenon of blood cells, thereby reducing the release of genomic DNA which can be mixed with free DNA, protecting the short fragments of plasma free DNA from further degradation, preventing the free DNA from cross-linking with proteins, and providing stable environment for the free DNA. The blood preservative for the free DNA of the invention has a good protective effect on the plasma freeDNA, can prevent genomic DNA contamination, and provides high quality samples for downstream free DNA detection.

The invention discloses a method for extracting total ribonucleic acid (RNA) from bracts and inflorescences of Anthurium andraeanum. The method comprises the following steps of: grinding young and tender bracts and inflorescences of the Anthurium andraeanum by using liquid nitrogen, standing an RNA extracting solution in a water bath at the temperature of 63 to 67 DEG C for 5 to 8min, adding chloroform A in an amount which is one fifth of the volume of the RNA extracting solution, violently shaking for 15 to 20s again, standing for 5 to 8min at room temperature, centrifuging to obtain a supernatant, treating the supernatant by using chloroform and a 8M LiCl solution, and centrifuging to obtain the total RNA of the bracts and the inflorescences of the Anthurium andraeanum. By the method, the using amount of toxic and harmful medicines is furthest reduced, and the hazard of RNA extraction on human bodies is reduced; high-quality RNA which is adequate and pure and is not subjected to deoxyribonucleic acid (DNA) contamination can be obtained; and the method is easy to operate, and a high-quality product is obtained in a short time.

Disclosed is a method for diagnosing cancer with high accuracy through quantification of cancer cell-derived DNA, which comprises the steps of: (1) extracting free DNA from a plasma collected from a test subject; (2) quantifying the extracted free DNA and calculating the free DNA content per unit volume of the plasma to obtain a first calculation value; (3) comparing the first calculation value with a second threshold value which is equal to or higher than a first threshold value; and (4) determining that the test subject is highly unlikely affected by cancer if the first calculation value is lower than the first threshold value, determining that the test subject is likely affected by cancer if the first calculation value is equal to or higher than the first threshold value and lower than the second threshold value, or determining that the plasma used for the quantification is contaminated by normal cell-derived DNA if the first calculation value is equal to or higher than the second threshold value.

The invention belongs to the technical field of gene expression analysis and discloses a method for detecting whether genome DNA contamination exists in a cDNA sample during watermelon gene expression analysis. The principle of the method is as follows: according to structure and sequence information of genes, forward and reverse primers are designed respectively on two adjacent exons, so that amplification products on a genome DNA template are larger than those on a cDNA template. According to the principle, specific primers of molecular chaperone DnaJ protein gene DNAJ widely existing in a watermelon are designed, the cDNA sample is taken as a template, PCR (polymerase chain reaction) amplification is performed by the aid of the primer pair, if amplification products larger than 87bp appear, the phenomenon indicates that the genome DNA contamination exists in the cDNA sample, and if only amplification products of 87bp appear, the phenomenon indicates that no genome DNA contamination exists in the cDNA sample. With the adoption of the method, the accuracy of watermelon gene expression analysis with technologies of real-time fluorescent quantitative PCR and the like can be further improved.

The invention discloses a microsatellite instability detection method, and belongs to the field of molecular diagnosis. The method used for detecting colorectal cancer microsatellite instability is invented based on multiplex fluorescent PCR technology and capillary electrophoresis technology. According to the method, a first primer pair represented by SEQ ID NO.1-2, a second primer pair represented by SEQ ID NO.3-4, a third primer pair represented by SEQ ID NO.5-6, a fourth primer pair represented by SEQ ID NO.7-8, and a fifth primer pair represented by SEQ ID NO.9-10 are used for multiplex fluorescent PCR and detection of five microsatellite points BAT-25, BAT-26, D2S123, D5S346, and D17S250. The sensitivity and the precision are higher; detection of internal reference microsatellite point Penta C is introduced to eliminate foreign DNA pollution in samples to be detected, and the credibility of obtained results is increased.

The invention relates to a zinc-copper superoxide dismutase SOD1 expression amount molecular detection method and a primer used in the method. The forward primer sequence is represented by the sequence 1, and the reverse primer sequence is represented by the sequence 2. According to the provided molecular detection method, a qPCR method is adopted for the first time, the content of SOD1 is detected in the gene level; at the same time, a set of targeting primer sequences and inner reference sequences is provided; even if the RNA extracted from a sample is polluted by DNA, the difference of expression amount of SOD1 gene in different human tissues and cells can be stably, specifically, and precisely detected, moreover, the repeatability is extremely high, and solved are the problems that the commonly-used enzyme activity detection kit can be badly influenced by artificial factors, and the stability and repeatability of the kit are both very bad.

The invention discloses a method for extracting and enriching a phage from aerobic granular sludge. The method comprises the following steps: (1) sludge grinding; (2) extracellular polymeric substancehydrolysis; (3) ultrasonic/vortex oscillation; (5) supernatant extraction; (5) secondary ultrafiltration concentration; and (6) sample preservation and fixation. The method is simple and convenient to operate, the reaction condition is mild, no toxic reagent is used, the problems that a granule sludge system is low in extraction efficiency due to EPS (extracellular polymeric substance) wrapping,and baterial cell lysis causes host DNA pollution are solved, and the method is an efficient and feasible phage extracting method. Compared with a traditional method for purifying a phage through combination of PEG and salting out, medicaments such as PEG and NaCl is not required to be used, the purifying time is shortened to about 6 h from orginal 24 h; and the obtained sample is low in salt concentration, further dialysis and desalting are not needed, and the obtained concentrated solution can be directly used for related operations such as a transmission electron microscope, DNA extraction,CsCl2 gradient centrifugation.

The invention discloses a reagent kit for detecting DNA (deoxyribonucleic acid) methylation. The reagent kit at least comprises biotin-labeled methylated lambda-DNA and streptomycin-enveloped magneticbeads. The invention further discloses application of the reagent kit to detecting methylation of a few DNA samples and a method for detecting the DNA methylation and application of the method. The low sample amount can reach 1 ng. According to the technical scheme, the reagent kit, the application and the method have the advantages that the lowermost initial sample amount required for detectingthe DNA methylation can be obviously lowered, specific reagent kits and experimental equipment can be omitted, accordingly, the cost can be saved, and the reagent kit and the method are high in universality; exogenous DNA pollution can be prevented when methylated DNA is enriched, influence of exogenous DNA information can be prevented if processes for detecting the DNA methylation by means of library construction and high-throughput sequencing are adopted, accordingly, the reagent kit and the method are favorable for further reducing the sequencing cost, and the quality of data can be improved.