30 results about "Lambda exonuclease" patented technology

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 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 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 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.