30 results about "Lambda exonuclease" patented technology

The invention discloses a preparation method for a long probe which can be used for multiplex ligation-dependent probe amplification (MLPA). The preparation method comprises the following steps: (1) selecting a vector which is irrelevant with a target sequence to be tested to serve as a template, and designing a primer according to the length of a target nucleotide sequence and the length of a target probe; (2) artificially synthesizing the primer; (3) performing polymerase chain reaction (PCR) amplification; (4) purifying and recovering PCR products; (5) digesting the PCR products by using Lambda exonuclease; and (6) recovering single-stranded deoxyribonucleic acid (ssDNA). The preparation method for the probe for the MLPA is low in cost, and all operations, such as the amplification, enzyme digestion and the like, can be finished only by a PCR instrument; recovery is performed by using a special ssDNA recovery kit after the enzyme digestion is finished, thereby, double-stranded DNA (dsDNA) and nucleotide which are not completely digested are eliminated, and the hybridization efficiency when an MLPA experiment is conducted is increased; and through full Lambda exonuclease digestion, the dsDNA can be changed into the ssDNA to the maximum degree, and thereby, the preparation efficiency of the probe is increased.

The invention discloses a rapid fluorescence detection method for 19# exon deleted mutation of a gene EGFR and an application. The method disclosed by the invention has the properties that by using Lambda exonuclease, an outstanding structure, of which 5' terminals are two bases, and a single strand labeled with fluorophore in DNA double strands can be rapidly hydrolyzed, and a DNA single strand with a 5' phosphorylated terminal in double-stranded DNA can be continuously hydrolyzed; and mutated base identification, PCR product single-stranding and fluorescence signal amplification processes are completed in a combined manner, so that entire detection flows are greatly simplified, the detection time is shortened, the relative detection limit is lowered to 0.01%, the sensitivity is high, andthus, the method has a very good application prospect in the aspects of clinical diagnosis, targeted medication instruction, postoperative accompanied detection and the like.

The invention discloses a gene mutation detection method based on selective elimination of wild strand background interference. A target sequence to be detected is amplified through PCR (Polymerase Chain Reaction) and then is processed into single-stranded DNA (Deoxyribonucleic Acid) through Lambda exonuclease; a thio-DNA strand which is complementary with a target region of a wild type DNA sequence and an RNA (Ribonucleic Acid) closed strand which is complementary with a non-target region are designed and synthesized; then the thio-DNA strand and the RNA closed strand are mixed with the single-stranded DNA, and DNase I is added to carry out cutting after temperature rising and annealing are carried out; a wild type DNA strand target region sequence is selectively cut off through the DNaseI under the guidance of the thio-DNA strand; a mutation type DNA strand is remained since mispairing exists in the target region and cannot be cut off by the DNase I, so that the abundance of the mutation type DNA strand is remarkably improved and the difficulty in detecting low-abundance gene mutation in the prior art is greatly reduced. The method disclosed by the invention does not need complicated and expensive instruments, is easy to operate and low in cost and can provides a result within 1 day; in-time and reliable gene mutation information can be provided for clinical early screeningof tumors and monitoring the recurrence after operation.

The invention relates to the technical field of nucleic acid detection material preparation, in particular to a preparation method and an application of a multi-fluorescent nucleic acid probe, 5-ethynyl-uracil deoxynucleotide (5-EdUTP) is used for replacing thymine deoxynucleotide (dTTP) for nucleic acid amplification, and high-density alkyne double-stranded DNA is obtained; after the obtained double-stranded DNA is treated by an excision enzyme Lambda Exonuclease, the 5'end phosphorylation labeled nucleic acid chain is digested, and the high-density alkyne single-stranded DNA is obtained; through a click reaction catalyzed by copper ions, a dye cy3-azide with an azide group is successfully modified to a single-chain DNA of high-density alkyne, and the multi-fluorescent nucleic acid probe is obtained. The fluorescent groups of the multi-fluorescent nucleic acid probe prepared by the invention are increased, signal amplification is realized structurally, the sensitivity of the fluorescent nucleic acid probe in researches such as analysis and detection, biological imaging and the like can be enhanced, and the practical value of the fluorescent nucleic acid probe is improved.

The invention discloses a rapid fluorescence detection method and application of exon 19 deletion mutation of EGFR gene. The method of the present invention utilizes Lambda exonuclease to rapidly hydrolyze the double-stranded DNA with a protruding structure of two bases at the 5' end and the single-strand marked with a fluorescent group, and to continuously hydrolyze the double-stranded DNA with 5' phosphate The nature of single-stranded DNA at the end, the combination of mutation base recognition, PCR product single-stranded and fluorescent signal amplification process greatly simplifies the entire detection process, reduces detection time, and the relative detection limit is as low as 0.01%. It has a good application prospect in clinical diagnosis, targeted drug guidance and postoperative accompanying detection.

The invention discloses an active determination method for Lambda exonuclease. The active determination method comprises the following steps: obtaining double-chain DNA with a phosphorylated 5' end on one chain, then reacting in a reaction system in the presence of the Lambda exonuclease by adopting the double-chain DNA as a substrate, determining the relative quantity of single-chain DNA or the double-chain DNA in the reaction system after the reaction is finished, and deducing the active degree of the Lambda exonuclease from a detection result. Compared with the method in the prior art, the active determination method has the advantages of no radioactive pollution, simplicity and fastness in operation and high sensitivity.

The invention relates to the preparation of oligonucleotide probes, in particular to a method for preparing long DNA probes containing multiple repeating units. Using blunt-end endonuclease and nicking endonuclease to simultaneously cut the plasmid containing multiple repeat unit DNA double strands, and then use the principle of lambda exonuclease to specifically cut, after cutting, separate by electrophoresis to obtain multiple repeat units Single-stranded DNA of interest for the unit. The method of the present invention is suitable for preparing DNA probes of all sequence components, especially for sequences that are difficult to perform PCR amplification, such as those containing multiple repeating units, and the length of the prepared probe can reach 800nt, solving the problem of multiple repeating unit DNA Technical issues in the preparation of long probes.

The invention belongs to the technical field of DNA sequence detection, and discloses a method for specifically regulating nuclease activity, designing corresponding Guiding chains for gene sequences at different mutation sites; synthesizing and preparing the required Guiding chains; Mix with the probe detection system so that the ratio of Guiding strand to substrate strand is 2:1; add Endo IV to the above mixed system to convert the substrate strand into a longer DNA double strand, and perform real-time fluorescence measurement. The regulation of endonuclease IV / APE-1 / lambda exonuclease activity involved in the present invention reduces the background signal due to nuclease side activity, and at the same time retains the main activity of nuclease cutting the target sequence to the greatest extent, thereby improving detection efficacy.

The invention discloses a preparation method for a long probe which can be used for multiplex ligation-dependent probe amplification (MLPA). The preparation method comprises the following steps: (1) selecting a vector which is irrelevant with a target sequence to be tested to serve as a template, and designing a primer according to the length of a target nucleotide sequence and the length of a target probe; (2) artificially synthesizing the primer; (3) performing polymerase chain reaction (PCR) amplification; (4) purifying and recovering PCR products; (5) digesting the PCR products by using Lambda exonuclease; and (6) recovering single-stranded deoxyribonucleic acid (ssDNA). The preparation method for the probe for the MLPA is low in cost, and all operations, such as the amplification, enzyme digestion and the like, can be finished only by a PCR instrument; recovery is performed by using a special ssDNA recovery kit after the enzyme digestion is finished, thereby, double-stranded DNA (dsDNA) and nucleotide which are not completely digested are eliminated, and the hybridization efficiency when an MLPA experiment is conducted is increased; and through full Lambda exonuclease digestion, the dsDNA can be changed into the ssDNA to the maximum degree, and thereby, the preparation efficiency of the probe is increased.

The invention discloses a preparation method of a modified DNA (deoxyribonucleic acid) hybridization probe for targeted hybrid capture. The preparation method mainly includes preparing a DNA template prior to PCR (polymerase chain reaction) amplification and purifying a PCR product; subjecting the purified PCR product to USER enzyme digestion and T4 DNA polymerase terminal blunting, wherein one of double-stranded DNA of an Lambda exonuclease hydrolysis product has 5'-phosphorylated single strand, so that bioti-labeled single-stranded DNA is obtained; purifying the bioti-labeled single-stranded DNA by a magnetic beads enrichment method to obtain the modified DNA hybridization probe. The preparation method of the modified DNA hybridization probe for targeted hybrid capture has the advantages that production cost is reduced greatly, and the preparation method is wide in application range including exon regions, intron regions, mitochondrion regions and the like of genomes of any species. The obtained DNA hybridization probe can be applied to construction and sequencing of next generation targeting libraries.

The invention discloses a method for detecting target miRNA based on G-quadruplex molecular beacon double-enzyme cascade isothermal amplification. The method comprises the following steps: taking a reaction system containing miRNA, a reaction buffer solution, Bsu DNA polymerase, Lambda exonuclease and G4MB, and incubating; detecting fluorescence intensity; and judging whether the miRNA contains the target miRNA or the content of the target miRNA according to the fluorescence intensity. The G4MB sequentially comprises a DNA fragment 1, a DNA fragment 2 and a DNA fragment 3 from the 5' end to the 3' end; the nucleotide sequence of the DNA fragment 2 is reversely complementary with the nucleotide sequence of the target miRNA; and one end of the G4MB is modified with a fluorescence marker anda phosphate group, and the other end of the G4MB is modified with a quenching group. Experiments prove that the method provided by the invention can be used for simultaneously detecting multiple target miRNAs, and is high in accuracy, high in sensitivity and good in specificity. The method disclosed by the invention has important application values.

The invention discloses a manganese dioxide sheet mimic enzyme sensor and a preparation method thereof as well as a T4PNK detection method. The preparation method comprises the following steps: (1) mixing a MnO2 nanosheet solution with a TMB (3,3',5,5'-tetramethyl benzidine) solution to obtain a MnO2-TMB mixed solution; (2) dissolving hairpin DNA in a Tris-HCl buffer solution, and adding ATP (adenosine triphosphate) and lambda EXO (lambda exonuclease) for mixing to obtain a DNA solution; and (3) mixing the MnO2-TMB mixed solution and the DNA solution to obtain the manganese dioxide sheet mimic enzyme sensor. The preparation method is simple in operation, and the sensor has the characteristics of high sensitivity, low detection limit and simple operation when used for detecting T4PNK.

The invention relates to a method for simulating recombination and non-trace cloning, which is characterized in that when a carrier primer is designed, a fragment of DNA basic group having a same sequence with a target gene is respectively added at 5' terminal of the primer, the obtained carrier DNA terminal and the inner part of the target gene have a coupling area, the carrier DNA and the target gene are mixed, and Lambda Exonuclease and T4 DNA polymerase are used for treatment to obtain recombinant DNA. The method for simulating recombination and non-trace cloning has the following beneficial effects that 1) the target gene amplified through PCR is not required, the mutation caused by PCR is reduced; 2) the target fragment can be specifically cloned in a DNA mixture; and a trace of an enzyme site is not kept.

The invention discloses a set of oligonucleotide aptamers capable of specifically recognizing streptococcus agalactiae, and belongs to the field of food hygiene and clinical medicine detection. Aiming at the defects that no oligonucleotide aptamers for the streptococcus agalactiae are in the prior art, the oligonucleotide aptamers provided by the invention combine an SELEX technology with the streptococcus agalactiae, a single stranded secondary library is prepared by incubating, cleaning, dissociating, amplifying, and digesting by using lambda exonuclease again and again for 12 rounds, the oligonucleotide aptamers capable of specifically combining with the streptococcus agalactiae are selected from a single stranded DNA random library; and three aptamers with the best effect are obtained by sequencing, affinity analysis and specificity analysis, wherein the nucleotide sequences are selected from 1 to 3 in the sequence table. The set of the oligonucleotide aptamers provide specific and efficient recognition ligands for analyzing and detecting the streptococcus agalactiae in the food hygiene and clinical blood samples and novel choices for developing and replacing a conventional method that detects the streptococcus agalactiae depending on antibodies.

The invention belongs to the technical field of nucleic acid probes and provides an oligonucleotide probe. The probe consists of a sequence complementary to the target sequence, a G-quadruplex blocking sequence and a G-quadruplex sequence from the 5'-3' end. The G-quadruplex blocking sequence is complementary to part of the G-quadruplex sequence, forming a stable hairpin structure, and enclosing the G-quadruplex sequence inside the probe. When detecting target molecules, the probe first recognizes the target sequence and forms a double-stranded structure, then under the digestion of Lambda exonuclease, the hairpin structure is opened, the G-quadruplex sequence is released, and the cycle repeats, and finally the trace target Molecular information is converted into a large number of G-quadruplex sequences, and then converted into readable optical, electrical and other signals through the G-quadruplex sequences. The probe has the advantages of good stability, low cost, easy preparation, high throughput, high sensitivity and specificity, can realize the direct detection of single-stranded nucleic acid, and can also detect double-stranded nucleic acid and other molecules for indirect detection.

The invention relates to a method for sensitively detecting alkaline phosphatase through dual-signal amplification mediated by dephosphorylation-triggered transcription reaction. A 5'-phosphorylated T7 promoter single-chain is designed, and alkaline phosphatase can catalyze The 5'-phosphorylated T7 promoter is dephosphorylated, protecting the T7 promoter chain from digestion by lambda exonuclease, and the remaining T7 promoter can activate the transcription reaction mediated by T7 RNA polymerase, resulting in a large amount of RNA transcripts . Subsequently, these RNA transcripts are complementary paired with Taqman probes to form RNA-DNA duplexes, and duplex-specific nucleases are introduced to trigger cyclic cleavage of Taqman probes, resulting in significantly enhanced fluorescent signals. The method is simple to operate, has high sensitivity and high specificity, and can be applied to the screening of target inhibitors in complex biological samples and the quantitative detection of targets in cervical cancer cells.

The invention belongs to the field of biotechnology, and in particular relates to a method for measuring 8-oxoguanine DNA glycosylase based on a background signal suppression probe, a kit and an application thereof. The present invention utilizes the selectivity of Lambda exonuclease to the substrate structure and the recognition and cleavage of 8-oxoguanine by 8-oxoguanine DNA glycosylase, combined with the background signal to suppress the effect of the probe on the side activity of λexo Inhibition, a new 8‑oxoguanine DNA glycosylase fluorescence assay method was constructed. Specifically include: mixing λexo buffer, reporter probe sequences containing fluorescent groups and quenching groups, and background signal suppression probe sequences containing 8-oxoguanine; heating the mixture to 85°C, and then cooling down gradually Add the sample to be tested at 37°C; incubate at 37°C; after the incubation, add λexo to carry out enzyme digestion reaction, and then perform fluorescence detection.