10 results about "Upstream and downstream (DNA)" patented technology

A sequence for forming an editing substrate together with a target RNA editing site. The sequence and the upstream and downstream portions of the target RNA editing site form a specific double-chain secondary structure, so that efficient editing of the target RNA editing site is realized.

The present application relates to a primer set for detecting multiple mutations of human embryo beta-thalassemia, a kit and application thereof. The primer set comprises five primers for amplifying HBB a plurality of mutations in the gene region: HBB-F1, HBB-F2, HBB-F3, HBB-R1 and HBB-R2, the sequences of which are shown in SEQ ID NO: 1-5 respectively; and eight primers for amplifying HBB linkage SNP sites within 1Mb range of the upstream and downstream genes: L1-F, L1-R, L2-F, L2-R, L3-F, L3-R, L4-F and L4-R, the sequences of which are shown in SEQ ID NO: 6-13 respectively. Based on the specific combination of long fragment PCR amplification and long fragment high-throughput sequencing, the present application can simultaneously detect multiple mutations of beta-thalassemia in multiple embryo samples with high specificity, accuracy and speed.

This invention discloses a method based on SOD The primers and methods for gene screening of heat-resistant scallop varieties were developed using scallop cDNA and DNA as templates. PCR amplification was performed using upstream and downstream primers (SEQ ID No. 3 and SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6) to obtain samples containing scallop cDNA and DNA. SOD Gene sequence fragments from the exon and promoter regions were used as molecular markers. The genotypes were CT at the c.229 C>T site, CA at the c.284 C>A site, and CA at the c.348 C>A site in the exon region, and CA at the g.1124 A>C site and AT at the g.1359 T>A site in the promoter region. This method screened for high-temperature resistant scallop varieties, demonstrating advantages such as genetic stability, accuracy, and efficiency.

This article discloses a method for identifying the nature of thyroid nodules, including detecting the DNA methylation level in a sample selected from the following regions (1) and (2): (1) fragments of one or more of the following genes: COL23A1, ILDR2, DHRS3, KIF1A, GDNF, TBX18, and (2) nucleic acid regions within 10 kb upstream and downstream of the genes (1).

This invention belongs to the field of molecular biology technology, specifically relating to a kit, method, and application for detecting SNP sites. The kit includes: primers, including upstream and downstream primers for amplifying upstream and downstream sequence fragments of the SNP site; probes, including multiple probes that specifically recognize SNP site polymorphisms; multiple different first detection markers, each used to bind to one of the probes; and a second detection marker for binding to the upstream and downstream sequence fragments. By using patterned first detection markers to bind probes for differentiation, a large number of different probes can be mixed in the same reaction well. After the probe captures an amplified fragment containing a specific SNP site, it forms a first detection marker-amplified fragment-second detection marker complex with the second detection marker bound to the amplified fragment. Thus, the genotype of the SNP site can be distinguished based on the combination of the first and second detection markers, ultimately achieving simultaneous and efficient multiplex detection.

This article discloses a method for identifying the nature of thyroid nodules, including detecting the DNA methylation level in a sample selected from the following regions (1) and (2): (1) fragments of one or more of the following genes: GAS6, SOX17, ZMIZ1, TSHR, CDH1, MCRIP2, LINC01977, EGR3, and (2) nucleic acid regions within 10 kb upstream and downstream of the genes (1).

The application discloses a method for constructing a directional SNP mutant in Neurospora crassa, and the method comprises the following steps: designing and synthesizing a forward primer containing a target SNP and a reverse primer located downstream of a 3'-UTR of a gene, and amplifying a gene segment A containing a mutation site from genomic DNA as a template; respectively amplifying homologous arm segments B and C located upstream and downstream of the segment A, wherein a reverse primer of the segment B comprises a reverse complementary sequence of a forward primer of the segment A; amplifying a hygromycin B resistance gene from a plasmid pCSN44 as a template hph , wherein primers at two ends of the hygromycin B resistance gene are respectively reverse complementary to the reverse primer of the segment A and the forward primer of the segment C; fusing the segments B, A, hph and C into a fusion segment B-A-hph-C through fusion PCR; transforming the fusion segment into the Neurospora crassa through electroporation, and obtaining an SNP site-directed mutant strain through hygromycin resistance screening and first-generation sequencing verification. The application has the advantages of simple operation, high mutation efficiency and accurate site, and provides a reliable technical means for gene function research of the Neurospora crassa.

This invention belongs to the field of gene editing technology, specifically relating to a scarless gene editing system for *Sphingomonas* and its application. The scarless gene editing system comprises a first inducing plasmid, a donor plasmid, and a second inducing plasmid. The first inducing plasmid includes the following elements: the EXO gene, the Gam gene, the Beta gene, a tetracycline resistance gene, and replicon 3233. The donor plasmid includes an exogenous DNA fragment for homologous recombination capable of amplifying upstream and downstream homologous arms of the target gene, an FRT fragment, and a chloramphenicol resistance gene. The second inducing plasmid includes the following elements: the FLP gene, a bleomycin resistance gene, and replicon 3233. Based on this scarless gene editing system, this invention knocks out the *Sphingomonas* 1535 gene, achieving an engineered strain capable of producing zeaxanthin, and further knocks out the 516 gene to increase zeaxanthin yield.

The disclosed invention relates to a linear double stranded DNA product with enhanced exonuclease resistance due to the presence of protected nucleotides, preferably phosphorothioated nucleotides. The linear double stranded DNA comprises: a sense and antisense strand, at least two protected nucleotides at internal positions in the antisense strand, a single cassette comprising a coding sequence, and wherein the antisense strand has a protected nucleotide upstream and downstream of the cassette. By flanking the cassette, the protected nucleotides protect the cassette from exonuclease degradation. In preferred embodiments, the cassette comprises a promoter, and the sense strand also comprises protected nucleotides upstream and downstream of the cassette. A method of manufacturing the DNA product is disclosed, comprising rolling circle amplification (RCA) of a template in the presence of phosphorothioated nucleotides thereby inserting said nucleotides into the product in a sequence and concentration dependent manner, and restriction digesting the RCA product such that each fragment comprises a single cassette. The DNA products of the invention are transfected into cells, resulting in expression of recombinant GFP, and also used for in vitro transcription (IVT) reactions.

This invention belongs to the field of edible fungi genetic breeding technology, and relates to a Cordyceps militaris strain that does not produce aspergillin, with the preservation number CGMCCNo.40266. This strain does not contain the CCM_02059 gene, CCM_02060 gene, or any of the CCM_02059 to CCM_02060 genes. It also relates to a method for creating this strain through CRISPR / Cas9 gene editing, comprising the following steps: S1, determining and synthesizing the sgRNA nucleotide sequence of the target gene; S2, determining and synthesizing the nucleotide sequences of the upstream and downstream homologous arms of the target gene; S3, ligating the sgRNA nucleotide sequence described in S1 and the homologous arm nucleotide sequences described in S2 into the pAMA1-Cas9-hyg vector to construct a knockout vector; and S4, transforming the knockout vector described in S3 into Cordyceps militaris protoplasts via PEG-mediated transformation to obtain a Cordyceps militaris strain with the target gene knocked out. This invention utilizes CRISPR / Cas9 editing technology to edit the gene cluster of inoculin synthesis in Cordyceps militaris, disrupting the inoculin synthesis pathway and breeding Cordyceps militaris strains that do not produce inoculin and have no foreign gene insertions, which is of great significance to the development of the Cordyceps militaris industry.