55 results about "Nuclease digestion" patented technology

The invention provides methods and compositions for selectively amplifying one or more target polynucleotides in a sample. In one aspect, a plurality of selection oligonucleotides are provided that are capable of simultaneously annealing to separate regions of a target polynucleotide to form a complex that is enzymatically converted into a closed double stranded DNA circle that incorporates the sequence region between the two separate regions. Sequences that fail to form such complexes may be removed by nuclease digestion and the sequences of the remaining DNA circles may be amplified by a variety of techniques, such as rolling circle replication after nicking, PCR amplification after linearization, or the like.

The invention discloses a method for building a helicobacter pylori nucleic acid fingerprint spectrum, which comprises PCR (polymerase chain reaction) amplification, SAP (severe acute pancreatitis) enzymatic digestion, transcription and nuclease digestion, purification, mass spectrometer detection and the like. A helicobacter pylori nucleic acid fingerprint spectrum database is set up on the basis of the method. According to the generated mass peak spectrum of the experiment, the helicobacter pylori of a sample to be detected can be quickly identified, so the method can be widely applied in the fields of helicobacter pylori types and classification, environmental sanitation, public safety qunarantine and the like.

The invention discloses a DNA extraction method of Duck Enteritis Virus genome and the genome sequence. Firstly, the pyrolysis of chicken embryo fibroblast which is infected with DEV is processed by hypertonic buffer; then the nuclease is added for the digestion of cell DNA; and the virus genome DNA is obtained through phenol chloroform extracting method. The obtained sample is broken randomly into 2kb to 2.5kb fragments; the pUC19 vector is linked to transform host E. coli and construct genomic library. The positive clones in the library are randomly sequenced; the total sequence length has 6 times coverage rate for the virus genome. The sequence result is assembled and clustered to obtain the whole DEV genome sequence with the length of156512bp. The analysis shows that the genome structure is the same with that of the members of Varicella virus in Alpha- herpesus subfamily, which provides theoretical basis for the classification of DEV.

The invention provides a method for generating a polynucleotide sequence or population of sequences from parent single stranded polynucleotide sequences encoding one or more protein motifs, comprising the steps of

• • (a) providing single stranded DNA constituting plus and minus strands of parent polynucleotide sequences;
  • (b) digesting the single stranded polynucleotide sequences with a nuclease other than DNase I to generate populations of single stranded fragments;
  • (c) contacting said fragments generated from the plus strands with fragments generated from the minus strands and optionally, adding primer sequences that anneal to the 3′ and 5′ ends of at least one of the parent polynucleotides under annealing conditions;
  • (d) amplifying the fragments that anneal to each other to generate at least one polynucleotide sequence encoding one or more protein motifs having altered characteristics as compared to the one or more protein motifs encoded by said parent polynucleotides.

The present invention relates to a method for in vitro evolution of protein function. In particular, the method relates to the shuffling of nucleotide segments obtained from exonuclease digestion. The present inventors have shown that polynucleotide fragments derived from a parent polynucleotide sequence digested with an exonuclease can be combined to generate a polynucleotide sequence which encodes for a polypeptide having desired characteristics. This method may be usefully applied to the generation of new proteins (e.g., antibodies and enzymes) or parts thereof having modified characteristics as compared to the parent protein.

A method for detecting transcription factor protein by nucleic acid probe with nuclease protection PRET label includes preparing fluorescence labeled nucleic acid probe, carrying out combination reaction of nucleic acid probe with transcription factor protein, adding nuclease in combination reaction system to carry out enzyme nick reaction and detecting reaction system by fluorescence to reflect expression and activation level of transcription factor protein.

The preparation process of reovirus antigen subunit vaccine for grass carp includes the following steps: proliferation of GCRV873, which is the Hunan Shaoyang strain of reovirus, in grass carp kidney cell line; centrifugal purification and concentration of GCRV873 infected cell virus suspension; processing purified virus with surfactant to disintegrate complete virus structure to form viral nucleic acid and capsid protein subunit component; digesting with nuclease to degrade free virus genome dsRNA; dialysis to obtain reovirus protein subunit antigen preparation for grass carp containing no nuclease; and diluting with physiological saline to obtain the said vaccine. The vaccine of the present invention has the advantages of containing complete virus protein antigen component, containing no genetic virus matter RNA, simple preparation process, high product purity, etc.

The invention relates to a method of isolating antibacterial peptides and the isolated peptides thereof. The method comprises: constructing a fusion DNA sequence of yeast alpha mating factor leader sequence and a random peptide, inserting the sequence into a saccharomyces cerevisiae vector which contains an inducible promotor, digesting the unlinked linear DNA with exonuclease III and mungbean sprout nuclease, electrotransforming the yeast vector after purification, culturing the transformant 24h and covering the transformant with the upper medium which contains E. coli K12D31, and detecting the bacterial inhibition ring and the bacterial inhibition rate after inducing the expression by liquid medium, selecting the positive clones and amplifying by PCR, and determining the sequence. The method of the present invention can be used easily and effectively to isolate antibacterial oligopeptides and polypeptides and can be used as an alternative way to find antibiotics.

This disclosure teaches high throughput mutation screening methods allowing simultaneous analysis of multiple genetic regions of interest and sensitive detection of very low frequency mutation(s) by the use of a universalized approach. Methods comprise treating RNA:DNA heteroduplexes of interest with a ribonuclease, sequence extension by an RNA-primed DNA polymerase, ligation with a blocking adapter, and differential sequence fill-in followed by single-strand-specific nuclease digestion to permit full-length sequence extension and subsequent ligation with a tagged reporter adapter solely in mutants filled in with a complementary deoxyribonucleotide triphosphate. By forming tagged mutant-dual adapter hybrids or mutant-triple adapter hybrids, the detection and/or quantification of mutants may be directed to the commonly shared tag(s) or flanking adapter sequences for signal detection/enhancement or sequence amplification in all different mutants regardless of the source or the number of mutations involved, thereby avoiding the tremendous effort of multiple target-specific sequence amplifications. Methods may be performed wholly or partially in solution, on solid phase media, in large scale, adapted for automated or semi-automated analysis, and any combinations thereof.

A method for identifying aptamers that bind to target molecules may include contacting an oligonucleotide library with target molecule and digesting unbound oligonucleotides with one or more endonucleases, one or more exonucleases, or one or more endonucleases in combination with one or more exonucleases. The unbound oligonucletides lend themselves to nuclease digestion because their flanking sequences are complementary and form predictable, terminal, secondary structure (a Lariat Aptamer). Bound molecules (aptamers) are protected from nuclease digestion and become enriched. A method for identifying aptamers may further include optionally subjecting selected aptamers to one or more rounds of selection under conditions of increased stringency. A method for identifying aptamers may include yet further amplifying selected aptamers. The described methods may be performed in a screen for identifying aptamers either alone or in combination with other methods typically employed in the art for selecting aptamers (such as, e.g., SELEX). Also contemplated herein are systems and kits for accomplishing the above.

The invention discloses a method for preparing bacteria 16S rDNA fingerprint spectrums. The method comprises the steps of PCR (polymerase chain reaction) amplification, SAP (super absorbent polymer) enzymatic digestion, transcription and nuclease digestion, purification, mass spectrometer detection and the like. A nucleic acid fingerprint spectrum database of common bacteria is created based on the method. According to the mass spectrum peak chart generated through experiments, the bacteria to be detected can be classified and identified and the results can be widely applied to the fields of bacteria classification and identification, genetic evolution analysis, drug resistance screening application, import and export inspection and the like.

The invention discloses a method for nucleic acid sequencing through adopting nuclease digestion reaction. The method includes the following steps: (1), performing digestion and degradation on a nucleic acid target sequence through an enzyme, so as to generate an enzyme-digested product; (2), detecting the enzyme-digested product, and finally obtaining information of the nucleotide sequence. The method provided by the technical scheme is used for cutting the target sequence and determining an unknown sequence through the exonuclease, and is realized through utilizing the biodegradability of the exonuclease and sequencing the enzyme-digested product generated from degradation, so as to determine the enzyme-digested product.

There are disclosed compositions and methods to render nucleic acids resistant to nuclease digestion while maintaining sequence selective hybridization competency. The approach relies on utilizing nucleic acids as the polar head group of a nucleic acid-polymer amphiphile in order to assemble well-defined, discrete micellar nanoparticles. Dense packing of nucleic acid in the micelle corona allows for hybridization of complementary oligonucleotides while prohibiting enzymatic degradation.

The invention relates to a method for extracting and enriching phage in soil, which comprises the following steps: mixing fresh soil with a potassium citrate buffer solution according to a certain ratio, incubating the materials at low temperature for a period of time, and carrying out ultrasonic oscillation treatment on an extracting solution to extract the phage in soil from soil particles, centrifuging soil turbid liquid for multiple times to obtain a supernatant, sequentially filtering and degerming by using a sterilization filter membrane, reducing the volume of a filtrate by using a tangential flow filtration/ultrafiltration technology, and enriching bacteriophage; recovering bacteriophage in a polyethylene glycol precipitation manner, performing standing overnight at a low temperature, centrifuging the next day, dissolving the bacteriophage in a TE buffer solution, filtering the solution by using a filter membrane again for sterilization, and digesting impurities DNA and RNA ina sample by using nuclease; and finally performing subsequent DNA extraction, sample fixation and other operations on the obtained concentrated solution.

The invention discloses a method for preparing bacteria nucleic acid fingerprint spectrums. The method comprises the steps of PCR (polymerase chain reaction) amplification, SAP (super absorbent polymer) enzymatic digestion, transcription and nuclease digestion, purification, mass spectrometer detection and the like. A nucleic acid fingerprint spectrum database of common bacteria is created based on the method. According to the mass spectrum peak chart generated through experiments, the bacteria to be detected can be classified and identified and the results can be widely applied to the fields of bacteria classification and identification, genetic evolution analysis, drug resistance screening application, import and export inspection and the like.

The invention provides methods and materials for generating a reference library of restriction fragments from pooled nucleic acids that contain a sequence polymorphism. Preferably, such a library is formed by digesting genomic DNA from a pool of individuals with a first and a second restriction endonuclease to form a population of restriction fragments; isolating restriction fragments of the population digested by both the first and second restriction endonucleases and forming a first single stranded fragment population therefrom; separately isolating restriction fragments from the population digested by the first restriction endonuclease but not the second restriction endonuclease and forming a second single stranded fragment population therefrom; hybridizing the first and second single stranded fragment populations to form a population of duplexes; and isolating the population of duplexes to form a reference library of restriction fragments that contain sequence polymorphism. An important aspect of the invention is the use of the reference population of restriction fragments to compare the frequencies of polymorphic sequences between different population pools. Such comparisons may be accomplished by competively hybridizing DNA from the respective pools which has been enriched for the presence of a restriction site polymorphism with DNA from the reference population. Preferably, such competitive hybridization reactions are carried out the reference library attached to one or more solid phase supports. Most preferably, members of the reference library are attached to individual microparticles so that each microparticle has a unique fragment attached. After competitive hybridization, the microparticles may be analyzed and sorted for identifying those microparticles carrying sequences for which the pools being compared exhibit different polymorphic frequencies.

The invention provides a method for locating damage and modification sites on genomic DNA. Firstly, a damage or modification site of a DNA sample is converted into a breakpoint. Then thionucleotide and DNA polymerase I were added for incision translation. The sample obtained after the reaction is terminated is added with a nuclease digestion template sensitive to phosphorothioylation modificationDNA. Finally, the phosphorothioyl modified DNA was sequenced, and the 5'end of the obtained sequence was the original damaged or modified site. The method provided by the invention, the invention fills in the blank that the existing technology at home and abroad can not accurately locate the damage site on the genomic DNA, and can realize the quantitative and positioning analysis of the damage andmodification sites of DNA samples with different lengths and different sources. The invention is simple to use, easy to operate, and has no special requirements on the samples, high accuracy, low detection background influence and high resolution.

The invention relates to a method for identifying the type and site of a RNA binding with protein in a plant. The method mainly comprises the following steps: immobilizing the in-vivo binding state of protein and nucleic acid through ultraviolet crosslinking; subjecting immunoprecipitated RNAs to digestion with nuclease into small fragments; removing non-specifically bound RNAs by using SDS-PAGE and transferring methods; and carrying out reverse-transcription PCR amplification and large-scale sequencing by using adaptors connected with two ends of the RNA. The method has the advantages that 1) ultraviolet crosslinking is carried out in vivo, so real in-vivo binding conditions can be reflected; 2) since a covalent bond formed by ultraviolet crosslinking is irreversible, the length of nucleic acid binding with protein can be reduced through digestion by nuclease, and subsequent multi-step purification can greatly improve a ratio of signal to noise; 3) most non-specifically bound RNAs in a precipitation sample are removed through SDS-PAGE separation and subsequent transferring process removes free RNAs; and 4) the two ends of the RNA are connected with the adaptors, which makes subsequent PCR amplification and large-scale sequencing to be possible.

The invention discloses a differential nuclease digestion method and application thereof to detecting mRNA internal modification through LC-MS. The differential nuclease enzyme-digestion method comprises, according to the characteristic of difference of S1 nuclease and phosphodiesterase I in digestion of the 5'-end cap structure of mRNA, digesting the mRNA into nucleosides through the S1 nuclease and the phosphodiesterase I, then performing LC-MS detecting analysis, and combining the detecting results of digestion through the two nucleases to differentiate N7-methyguanosine (m7G) in the cap and the inside of the mRNA and according to the achieve qualitative and quantitative analysis on the m7G inside the mRNA. The differential nuclease enzyme-digestion method is high in sensitivity and selectivity, can quantitatively detect the content of m7G inside encaryotic mRNA and further can be applied to study of related functions of the m7G inside the mRNA.