30 results about "Uracil in DNA" patented technology

The invention discloses a specific marking method for 5-hydroxymethyluracil on DNA (deoxyribonucleic acid) and belongs to the technical field of molecular biology. The specific marking method comprises the following steps: transferring gamma-thiophosphate to 5-hmU from 5-O-thiotriphosadenine by using 5-hmU separated from bacteriophage M6 of pseudomonas aeruginosa so as to generate 5-thiophosphoricacid methyluracil, representing the generated 5-thiophosphoric acid methyluracil by using a liquid chromatogram-tandem mass spectrometry method, and marking sulfydryl through crosslinking chemistry,so as to achieve specific marking on 5-hydroxymethyluracil on DNA. By adopting the method disclosed by the invention, defects that a great amount of detection samples are needed, chemical reaction conditions are harsh, the reaction efficiency is low, and the like, can be effectively overcome.

Novel DNA constructs and host cells comprising the same are disclosed. DNA constructs comprise a transcription unit (e.g. operon) comprising DNA sequences encoding for ribonucleotide reductase and thioredoxin or a uridine kinase gene and/or a dCTP deaminase gene. In preferred embodiments the constructs comprising DNA sequences encoding for ribonucleotide reductase and thioredoxin further comprise DNA sequences encoding for thymidylate synthase and/or transcription units comprising sequences encoding for uridine kinase preferably together with dCTP deaminase. In particularly preferred embodiments, the host cells comprise constructs having all of the above characteristics wherein the host cell displays repressed or no uracil DNA glycosylase activity. This may be achieved by removal of the host cell ung gene. Use of host cells in the manufacture of pyrimidine deoxyribonucleotides e.g. thymidine is also disclosed.

The invention discloses a detection method of microRNA (micro Ribonucleic Acid), and belongs to the field of optical biosensing techniques. A to-be-detected sample is mixed with template DNA (Deoxyribonucleic Acid), an obtained first mixture is annealed and hybridized, a buffer solution containing polymerase Klenow fragment, deoxyuracil nucleoside triphosphate and terminal deoxynucleotidyl transferase is added into a reaction product, an obtained second mixture is caused to react for 4h at 35 DEG C in a water bath, so as to prepare a polyuracil base sequence; sodium ascorbate is added into a polyuracil base sequence solution obtained in the step (4), and then CuCl2 is added into an obtained third mixture, and an obtained fourth mixture is caused to react for 10min at a room temperature, so as to prepare a red-fluorescence copper nano cluster which uses the polyuracil base sequence as a template, thereby obtaining a solution containing the red-fluorescence copper nano cluster. The intensity of the red fluorescence of the solution including the red-fluorescence copper nano cluster is measured by adopting a spectrofluorophotometer; by comparing the result about the intensity of the red fluorescence with a standard relation curve, the concentration of the microRNA is calculated and obtained.

Disclosed herein is an efficient method of generating a library of variants of a sequence of interest, such as may be used in directed evolution, in one embodiment, the method includes an amplification reaction, e.g. error-prone PCR, to generate double-stranded DNA (dsDNA) variants of a sequence of interest, after which one strand of the dsDNA variants may be selectively degraded to produce single-stranded DNA (ssDNA) variants. The ssDNA variants may be hybridized to ssDNA intermediaries, e.g., uracilated circular ssDNA intermediaries, to form heteroduplex DNA, which may be transformed into cells, such as E. coli cells, yielding a library of variants. This method eliminates the inefficient sub-cloning steps and the need for costly primer sets required by many prior methods.

The invention discloses a preparation method and application of a DNA polymerase accelerant. The DNA polymerase accelerant is composed of three kinds of heat-stability protein which are uracil DNA glycosidase UDG, hypoxanthine DNA glycosidase AlkA and endonuclease IV respectively. UDG is in charge of cutting off dU basic groups generated in a PCR, AlkA is in charge of cutting off generated dI basic groups, and endonuclease IV is in charge of cutting off basic group removal loci. The joint action of the three kinds of protein can achieve the purposes that the inhibition effect of the dU/dI basic groups on the activity of B type DNA polymerase is released; the dU/dI basic groups are repaired and corrected, and the PCR faithfulness of the A type and B type DNA polymerase is improved. In addition, the activity of 3'-excision enzymes of endonuclease IV can provide deleted correction activity of the A type DNA polymerase. The DNA polymerase accelerant can effectively increase the PCR yield of the B type DNA polymerase by 1-3 times.

The invention belongs to the technical field of DNA (deoxyribonucleic acid) glycosylase detection and in particular relates to a fluorescent chemical sensor for synchronously detecting multiple DNA glycosylases and a detection method and application of the fluorescent chemical sensor. The invention provides a method for simultaneously detecting multiple DNA glycosylases, the detection precision can be improved, the analysis and detection cost can be reduced, and the clinical application practicability can be improved. The invention provides a method for simultaneously detecting human alkyl adenine DNA glycosylase (hAAG) and uracil DNA glycosylase (UDG), monomolecular detection and rolling circle amplification driven fluorescent molecules of different codes are combined, multiple DNA glycosylases in cancer cells can be simultaneously detected at a monomolecular level, and normal cells and cancer cells can be also distinguished. The fluorescent chemical sensor can be further applied to analysis on enzyme kinetic parameters and screening on DNA glycosylase inhibitors, and has great potential in biomedical research, clinical diagnosis and medicine development.

The invention discloses an RNA (Ribonucleic Acid) 4-mercaptouridine specific labeling method and application thereof, and belongs to the technical field of nucleic acid chemistry. According to the method, a structure similar to cytosine (C) is generated under a mild condition by utilizing a reaction mechanism that hydrazine hydrate is used for carrying out addition elimination on RNA 4-mercaptouridine; in the reverse transcription process of RNA, the original A: T pairing at the position of 4sU is changed into C: G pairing, so that the position of 4sU is subjected to single base recognition. The method is combined with a sequencing technology and can be used for detecting the position of naturally existing 4sU and researching the dynamic state of a transcriptome.

The present disclosure provides a method for the cost effective bisulfite-free identification of the position of one or more of 5-methylcytosine, 5-hydroxymethylcytosine, 5-carboxycytosine, and 5-formylcytosine in DNA, including whole genome DNA, and a method for the cost effective bisulfite-free identification of the position of one or more of 5-methylcytosine, 5-hydroxymethylcytosine, 5-carboxycytosine, and 5-formylcytosine. The methods described herein are based on the conversion of a modified cytosine (5mC, 5hmC, 5fC, 5caC) to a dihydrouracil (DHU), e.g. By a TET-assisted pyridine borane treatment, followed by cleavage of the DHU with an endonuclease and identification of a cleavage site corresponding to the position of the modified cytosine.

The present invention discloses a kit for guiding 5-fluorouracil medication. The invention provides a primer group, which comprises a primer pair A and a primer pair B, wherein the primer pair A comprises single-stranded DNA molecules represented by the sequence 1 and single-stranded DNA molecules represented by the sequence 2, and the primer pair B comprises single-stranded DNA molecules represented by the sequence 3 and single-stranded DNA molecules represented by the sequence 4. The present invention further provides the kit for identifying the IVSl4+lG>A point mutation of gene DPYD and the 665C>T point mutation of gene MTHFR, wherein the kit comprises the primer group. According to the present invention, the kit can be used for detecting the two SNP sites associated with the 5-fluorouracil sensitivity so as to be used to clinically guide the individualized use of the 5-fluorouracil, and improve the targeting property and the predictability of the clinical treatment.

The invention discloses uracil auxotrophic hansenula polymorpha and a preparation method and application thereof. The preparation method of the uracil auxotrophic hansenula polymorpha comprises the steps that orotidine-5'-phosphate decarboxylase genes in hansenula polymorpha original strains are knocked out, or the content of proteins coded by the orotidine-5'-phosphate decarboxylase genes are reduced, or the proteins coded by the orotidine-5'-phosphate decarboxylase genes are inactivated, and the uracil auxotrophic hansenula polymorpha is obtained, wherein knockout of the orotidine-5'-phosphate decarboxylase genes are conducted with the steps that M1, coding of the orotidine-5'-phosphate decarboxylase genes is early stopped; and M2, exogenous DNA fragments are inserted into the orotidine-5'-phosphate decarboxylase genes. The built uracil auxotrophic hansenula polymorpha is stable in heredity, has no obvious difference from wild type strains in other growth characteristics, and can be used as transformed chassis cells in metabolic engineering or synthetic biology, and cell factories of efficient expression recombinant proteins.

The invention discloses endonuclease and application thereof. An amino acid sequence of the endonuclease is shown as SEQ ID NO: 1 or SEQ ID NO: 2, or the amino acid sequence of the endonuclease and an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 have at least 70% of sequence identity. The endonuclease can be used for directly and specifically recognizing deoxyuracil (deoxyuridine) on a DNA strand and cutting a first phosphodiester bond located at a 3'-terminal of the deoxyuracil is cut, cutting can be independently completed without combined action with other endonucleases with AP site lyase activity, and the endonuclease has wide application prospects in the fields of in-vitro molecular diagnosis of DNA damage mutation and the like. In addition, the invention provides a cutting site, i.e., the first phosphodiester bond located at the 3'-terminal of the deoxyuracil for the first time, and the cutting site is not reported in the prior art.

The invention relates to a method for non-specifically amplifying natural short-fragment nucleic acid. The method comprises the following steps: (1) performing terminal repair on the natural short-fragment nucleic acid to obtain terminal-repaired nucleic acid; (2) connecting the terminal-repaired nucleic acid with a double-chain connector to obtain a connection product, wherein each chain of the double-chain connector only comprises three basic groups; (3) performing PCR amplification on the connection product by using a PCR primer with a deoxyuridine marker to obtain a PCR product, wherein the PCR primer is completely or partially complementary to one chain of the double-chain connector and only comprises three basic groups; and (4) performing enzyme digestion on the PCR product by usingan enzyme with a deoxyuridine cutting function, and performing enzyme digestion by using an enzyme with 5'->3' polymerase activity and 3'->5' exonuclease activity in the presence of a deoxynucleotidesolution to obtain a non-specific amplification product of the natural short-fragment nucleic acid, wherein the deoxynucleotide solution only comprises complementary basic groups of the basic groups which are lacked by the primer. The invention also relates to a kit for implementing the method.

The invention discloses a method for detecting uridine monophosphate modification of protein. The method comprises the following steps: by using biotin-labeled UTP (Biotin-16-UTP) as a raw material, under the action of uridine monophosphate transferase YdiU, the protein occurring UMP modification having biotin labeling; and subsequently, verifying whether the protein to be detected has a biotin label or not by using a biotin-streptavidin imprinting method. The method has the characteristics of simplicity, convenience, high sensitivity and the like, can be used for high-throughput screening ofproteins subjected to uridine monophosphate acidification in prokaryotic and eukaryotic cells, can determine modification sites in combination with mass spectrometry detection, and provides an effective identification and research means for research of protein uracil mononucleotide modification.