34 results about "HpaII" patented technology

The invention relates to a construction method and a fermenting method of antibiotic-resistance-free recombinant bacillus subtilis for expressing glutamate decarboxylase and belongs to the technical field of bioengineering. The construction method includes taking bacillus subtilis WB600 as an original strain, and knocking out a D-alanine racemase gene on a chromosome of the bacillus subtilis WB600 so as to obtain D-alanine deficient WB600 (dal); fusing an optimized gad gene with an antibiotic-resistance-free expression vector pUB110 (a antibiotics resistance gene is replaced by the D-alanine racemase gene) through an overlap extension PCR technology to obtain a polymer, transforming the polymer into competence of the bacillus subtilis WB600, and enabling the polymer to recombine in a host so as to obtain a recombinant plasmid Pub-HpaII-P43-gad-dal, which is named as bacillus subtilis SK44.001 with the preservation number being CCTCC NO:M 2016774. The fermentation liquor enzyme activity of the antibiotic-resistance-free recombinant bacillus subtilis can be up to 8.6 U/mL, and accordingly the antibiotic-resistance-free recombinant bacillus subtilis has significant industrial application value.

The invention discloses a method for detecting the single-nucleotide polymorphism of a goat ATBF1 gene and the application of the goat ATBF1 gene. According to the method, the goat whole-genome DNA to be tested is taken as a template, the goat ATBF1 gene is amplified by use of the PCR technology, the PCR product is digested by use of a restriction enzyme MspI or HpaII, next, the agarose gel electrophoresis is performed, and the genotype of No.25748nt or No.163517nt SNP of the goat ATBF1 gene is identified according to the electrophoresis result; different genotypes of the No.25748nt and No.163517nt SNPs of the ATBF1 gene have remarkable relevance with the growth and development traits of the goat, and therefore, the ATBF1 gene is advantageous for quickly establishing a good goat genetic resource population.

The invention discloses a novel promoter and application thereof, and belongs to the field of microbial genetic engineering. According to a method disclosed by the invention, the related promoter Pd(-35 to -10) HpaII gene is formed by series connection modification on the core area based on the promoter PHpaII; under control and transcription of the novel promoter Pd(-35 to -10) HpaII, the fibrinolytic activity of the recombinant nattokinase is up to 200.8 FU/ml. The method is efficient, safe and capable of effectively improving the expression quantity of nattokinase, and provides a theoretical basis for further study on influence of the promoter on heterologous gene expression.

The invention discloses a method for protecting efficient recombinant expression of restriction enzymes by virtue of methylase. The method is characterized in that the methylase is M.Sss I; and the restriction enzymes D are selected from AatII, AciI, AclI, AfeI, AgeI, AscI, AsiSI, AvaI, BceAI, BmgBI, BsaAI, BsaHI, BsiEI, BsiWI, BsmBI, BspDI, BspEI, BsrFI, BssHII, BstBI, BstUI, BtgZI, ClaI, EagI, FauI, FseI, FspI, HaeII, HgaI, HhaI, HinP1I, HpaII, Hpy99I, HpyCH4IV, KasI, MluI, NaeI, NarI, NgoMIV, NotI, NruI, Nt.BsmAI, Nt.CviPII, PaeR7I, PluTI, PmlI, PvuI, RsrII, SacII, SalI, SfoI, SgrAI, SmaI, SnaBI, TliI, TspMI, XhoI, XmaI and ZraI. According to the method provided by the invention, the broad-spectrum protective methylase M.Sss I is directly adopted in a recombinant engineering bacterium construction process, without conducting complex methylase gene screening on a single restriction enzyme, so that the method is broad in application scope and is capable of greatly simplifying a recombinant expression process.

The invention discloses a colorimetric sensing method for detecting activity of DNA transmethylase based on The DNA chain substituting circulation amplifying technology. The method is designed as that the neck to be recognized by CpG transmethylase M.SssI and HpaII restriction enzyme contains hairpin DNA I with the 5'-CCGG-3' sequence; the presence of M.SssI can be determined by determining whether the hairpin DNA I is methylated and whether the hairpin DNA can be cut by HpaII to cause gathering discoloration of AuNPs; the sensitive detection of M.SssI can be achieved by the colorimetric method.

The invention discloses a PCR detection method for identifying germplasms of four populations of Pelodiscus sinensis, a primer group and a kit. The primer group comprises primers of SEQ No.1/SEQ No.4, SEQ No.2/SEQ No.5 and SEQ No.3/SEQ No.6. The kit comprises a 2*TaqPCR premixed liquid, a 5U/mul enzyme BglI and 10*digestion buffer liquid A, a 5U/mul HpaII and 10*digestion buffer liquid B, a 5U/mul ClaI and 10*digestion buffer liquid C, 10mg/ml BSA, a contrast DNA template and a 10pM amplimer. The method comprises the following steps: carrying out PCR amplification of extracted DNA through using the primer group, digesting to obtain a digestion product, carrying out agarose gel electrophoresis, and comparing the obtained electrophoresis result with a standard electrophoretogram to obtain a result. The detection method has the advantages of simplicity, good reaction stability and repeatability, and cost saving.

The invention provides a human SGIP1, SCAND3 and MYO1G gene methylation detection kit. The human SGIP1, SCAND3 and MYO1G gene methylation detection kit comprises methylation-sensitive restriction enzymes, a digestion buffer solution and a fluorescent PCR reaction solution, and the methylation-sensitive restriction enzymes are AciI, BstUI and HpaII. The detection kit adopts an enzyme mixture of themethylation-sensitive restriction enzymes being AciI, BstUI and HpaII, the digestion efficiency can be effectively improved, incomplete digestion is avoided, the false positive rate is reduced, the sensitivity is good, and the human SGIP1, SCAND3 and MYO1G gene methylation detection kit is suitable for a variety of sample types such as plasma DNA, tissue DNA and cell DNA.

The invention pertains to the technical field of the preparation of a pig genetic marker, more particularly relates to a pig production trait gene MX1 eighth intron mutant site polymorphism detection method and the clone used as the genetic marker of the pig production trait, and the application. Genome DNA is extracted from pig blood, primer PCR amplification is designed, a 563bpDNA sequence is obtained by clone resequencing, and the mutation of A167-G167 is found at the position of 167bp of a sequence list SEQID NO: 1 by sequence comparison analysis, thus causing the polymorphism of HpaII-RFLP. By utilizing the genetic marker, the genotype frequency and the gene frequency of a marker gene and the association analysis of the pig production trait are detected in different swineries, showing that notable association exists between the genetic marker and certain important production trait of the pig. The invention also discloses an SNP typing detection technology of the eighth intron of the pig MX1 gene, thus providing a new marking and detection method for marker auxiliary selection of the pig.

The invention discloses a kit for identifying human methylene tetrahydrofolate reductase (MTHFR) gene polymorphism rs2274976 by using MspI. The kit comprises a forward primer and a reverse primer for amplifying sequences near a human MTHFR gene polymorphism rs2274976 locus, wherein the last base at the 3'end of the forward primer is adjacent to an rs2274976 polymorphic base, and the last second base of the forward primer is a mismatched base C, so that the forward primer and polymorphic G allele form a CCGG structure in an amplification product; and the forward primer is shown as SEQ ID NO:1 or SEQ NO:11, and the reverse primer is shown as SEQ ID NO:2, SEQ ID NO:7 or SEQ NO:12. The kit also comprises restriction enzyme MspI or HpaII, and an instruction book for implementing identification. The kit is easy to operate, low in cost, and wide in application range.

The invention belongs to the technical field of DNA methylation analysis, and discloses an electrochemical analysis method based on a tetrahedral DNA nanoprobe and application, and the electrochemical analysis method comprises a tetrahedral probe structure sequence, different DNA methylation target sequences, a signal amplification sequence and a comparison sequence. According to the present invention, the optimal performance is represented under the conditions of the tetrahedral probe fixation time of 3 h, the target sequence hybridization time of 1 h and the HpaII incision enzyme digestion time of 2 h, the good detection ability is represented within the range of 1 amol/L to 1 pmol/L, the detection limit reaches 0.93 amol/L, and the sensitivity is high; the electrochemical biosensing technology has the unique advantages in the aspect of DNA methylation detection: specific analysis on a methylated DNA target sequence can be completed within a short time, the operation is convenient, the price is low, the sensitivity is high, the specificity is good, the stability is strong, and the electrochemical biosensing technology can be effectively used for DNA methylation detection in complex systems such as serum.

The invention relates to a construction method for forest-tree genome DNA (deoxyribonucleic acid) methylation inheritance linkage maps, and belongs to the technical field of molecular genetics. The construction method includes the steps: 1) extracting genome DNA of xylem tissues; 2) respectively performing dual-enzyme digestion for the genome DNA by the aid of first group of EcoRI and HpaII enzyme and second group of EcoRI and MspI enzyme, connecting connectors on two groups of dual-enzyme digestion products to obtain two groups of enzyme digestion connecting products; sequentially performing pre-amplification and selective amplification by the aid of connecting amplification products, separating selective amplification products by the aid of capillary electrophoresis to obtain population genome methylation marker sites; 4) selecting population methylation polymorphic sites meeting Mendelian inheritance separation from the population genome methylation marker sites to construct the methylation inheritance linkage maps. The construction method is efficient, stable and easy to operate, so that forest-tree population epigenetics research progress can be accelerated.

The key enzyme genes, namely 3-phosphoglyceraldehyde dehydrogenase (gapdh) and 3-hydroxyl-2- butanone reductase (acr), which participate in the coenzyme cyclic regeneration process in the synthesis route of 2,3-butylene glycol, are cloned from B. amyloliquefaciens B10-127; then the genes are inoculated to a shuttle expression plasmid pMA5-HpaII so as to successfully establish a shuttle expression plasmid containing gene gapdh and gene acr pMA5-HpaII-acr-HapII-gapdh, and the pMA5-HpaII-acr-HapII-gapdh is transferred to bacillus amyloliquefaciens B10-127 so as to obtain bacillus amyloliquefaciens, which is strengthened in gapdh and acr gene expression, namely pMA5-HpaII-acr-HapII-gapdh/B. amyloliquefaciens. The 2,3-butylene glycol output of the recombinant bacterium is raised by 14.7% compared to that of the original bacterium, the byproduct contents of 3-hydroxyl-2-butone, lactic acid and butanedioic acid are reduced by 65.6%, 43.8% and 42.4, and moreover the fermentation period is shortened. Through the processes of material supplement and fermentation, the 2,3-butylene glycol output can reach 121.3 g/L.