Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We introduce a novel experimental strategy for DNA mutation detection named the Mismatch Identification DNA Analysis System (MIDAS) [1, 2], which has an associated isothermal probe amplification step to increase target DNA detection sensitivity to attomole levels. MIDAS exploits DNA glycosylases to remove the sugar moiety on one strand (the probe strand) at a DNA base pair mismatch. The resulting apyrimidinic/ apurinic (AP) site is cleaved by AP endonucleases/lyases either associated with the DNA glycosylase or externally added to the reaction mixture. MIDAS utilizes 32p- or FITC-labeled oligonucleotides as mutation probes. Generally between 20-50 nucleotides in length, the probe hybridizes to the target sequence at the reaction temperature. Mismatch repair enzymes (MREs) then cut the probe at the point of mismatch. Once the probe is cleaved, the fragments become thermally unstable and fall off the target, thereby allowing another full-length probe to hybridize. This oscillating process amplifies the signal (cleaved probe). Cleavage products can be detected by electrophoretic separation followed by autoradiography, or by laser-induced fluorescence-capillary electrophoresis (LIF-CE) of fluorophore-labeled probes in two minutes using a novel CE matrix. In the present experiments, we employed the mesophilic Escherichia coli enzyme deoxyinosine 3'-endonuclease (Endo V), and a novel thermostable T/G DNA glycosylase, TDG mismatch repair enzyme (TDG-MRE). MIDAS differentiated between a clinical sample BRCA 1 wild-type sequence and a BRCA1 185delAG mutation without the need for polymerase chain reaction (PCR). The combination of MIDAS with LIF-CE should make detection of known point mutations, deletions, and insertions a rapid and cost-effective technique well suited for automation.
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PMID:Mutation identification DNA analysis system (MIDAS) for detection of known mutations. 1038 Jul 53

The DNA fragment detection focusing technique has further enhanced the sensitivity and information of DNA targets. The DNA fragment detection method was established by capillary electrophoresis with laser-induced fluorescence detection and restriction endonuclease chromatographic fingerprinting (CE-LIF-REF) in our experiment. The silica capillary column was coated with short linear polyarclarylamide (SLPA) using nongel sieving technology. The excision product of various restricted enzymes of DNA fragments was obtained by REF with the molecular biology software Primer Premier 5. The PBR322/BsuRI DNA marker was used to establish the optimization method. The markers were focused electrophoretically and detected by CE-LIF. The results demonstrate that the CE-LIF-REF with SLPA can improve separation, sensitivity and speed of analysis. This technique may be applied to analysis of the excision product of various restricted enzymes of prokaryotic plasmid (pIRES2), eukaryote plasmid (pcDNA3.1) and the PCR product of codon 248 region of gastric cancer tissue. The results suggest that this method could very sensitively separate the excision products of various restricted enzymes at a much better resolution than the traditional agarose electrophoresis.
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PMID:Study on detection of mutation DNA fragment in gastric cancer by restriction endonuclease fingerprinting with capillary electrophoresis. 2176 17

The methylation of the promoter region of DNA is an important regulatory mechanism for the downstream gene expression, and the extent of methylation has been linked to cancer formation. In this study, we report a simple method to screen for the degree of DNA methylation by combined bisulfite restriction analysis (COBRA) and capillary electrophoresis with laser-induced fluorescence (CE-LIF). After treating genomic DNA with sodium bisulfite, nested-PCR amplification and endonuclease (Taq I) digestion were performed. The digested DNA fragments were then separated by capillary electrophoresis using 1.5% poly(ethylene) oxide (M(ave), 8,000,000 g/mol) in the presence of electroosmotic flow. The improvement for DNA amplification using the nested PCR described here corresponded to approximately ten cells. In addition, the level of DNA methylation shown in the electropherograms obtained corresponded to the original percentage of DNA methylation from commercial available standard sample (0-100%). The electrophoretic patterns demonstrated that the six cancer cell lines tested displayed different degrees of DNA methylation and could be differentiated by hierarchical cluster analysis. Furthermore, the DNA methylation level was eliminated after treating the cells with an anti-cancer drug (5'-aza-2'-deoxycytidine). Together, these results suggest that CE-LIF is a potentially useful and cost-effective tool for cancer diagnosis or prognosis based on the heterogeneity in a patient's DNA.
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PMID:Determination of the heterogeneity of DNA methylation by combined bisulfite restriction analysis and capillary electrophoresis with laser-induced fluorescence. 2234 9

Background: Gout arthritis is a common inflammatory arthritis and it poses a major threat to human health. Objective: A method of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for the detection of HSP60 gene polymorphism has been developed and applied to the exploration of correlation between gouty arthritis and HSP60 gene polymorphism. Methods: The genomic deoxyribonucleic acid from 59 patients with gouty arthritis and 64 control subjects was extracted and the conservative fragment of HSP60 was amplified. The products were digested with restriction endonuclease NlaIII and then separated and detected by the proposed method. Results: In the case group, there were 7 cases of TT genotype, 29 cases of CC genotype, and 23 cases of CT genotype. In the control group, there were 4 cases of TT genotype, 6 cases of CC genotype, and 54 cases of CT genotype. The detection results of the samples were statistically analyzed by binary logistic regression and Spearman correlation analysis. After adjusting gender, age, and other compounding factors, the TT genotype and CT genotype of the HSP60 gene were found to affect gouty arthritis. Conclusions: When used for gene polymorphism research, the proposed CE-LIF method has the advantages of high efficiency, rapidity, sensitivity, and low sample consumption. A moderate correlation between gouty arthritis and HSP60 genotype distribution was discovered for the first time. Highlights: A new method using CE-LIF for the detection of HSP60 gene polymorphism of 59 patients with gouty arthritis and 64 control subjects in China. The correlation between gouty arthritis and HSP60 gene polymorphism was explored for the first time.
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PMID:Capillary Electrophoresis with Laser Induced Fluorescence Detection for Study of the Association of HSP60 Gene Polymorphism with Gouty Arthritis. 3034 Jun 51