Restriction endonucleases digesting DNA in PCR buffer

Six commonly used restriction endonucleases (REs) (Acc I, Ban II, EcoR I, Hind III, Sac I, Sca I) were tested for their ability to directly digest DNA completely in the Polymerase Chain Reaction (PCR) buffers. The results showed that: with the requirement for addi- tional magnesium supplemented as activator, REs, except EcoR I appeared star activity, completely digested unmethylated lambda DNA af- ter overnight incubation in PCR buffer and functioned as equally well as in recommended Restriction Enzyme Buffer provided with each enzyme; all REs tested completely digested PCR products in PCR buffer, it implied digestion of PCR products may often be performed di- rectly in the PCR tube without the requirement for any precipitation or purification steps; and the concentration of MgCl2 from 2.5 mmol·L-1 to 10 mmol·L-1 did not significantly affect activity of REs in PCR buffer. This simplified method for RE digestion of PCR prod- ucts could have applications in restriction fragment length polymorphism (RFLP) analysis and single-stranded conformational polymor- phism (SSCP) analysis of large PCR products. However, usage of this procedure for cloning applications needs further data.

Human DNA contains sequences homologous to the 5'-non-coding region of hepatitis C virus: characterization with restriction endonucleases reveals individual varieties

Objective To investigate a 272 base pair section of the 5' non coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (HCV) negative human subjects (not patients) Mothods This sequence section bears interest because ① it harbors several potential methylation (Cp rich) sites, and ② it represents the largest part of its internal ribosomal entry site A pre PCR digestion protocol was established making consistent use of four restriction endonucleases selected for certain features: SmaI, XmaCI, MspI, and HpaII are inhibited if methylation(s) are present at certain cytosines within their cutting sequences Results The suspected HCV specific sequence was found in the DNA of each subject tested The pre PCR digestion assay reveals individual differences in their pattern of methylation, which may be due to possible epigenetic phenomena Conclusions The results provide formal proof that these HCV specific sequences are contained in the genomic or extra chromosomal target DNA, and probably belong to a new class of endogenous sequences

Inhibition of DNA restrictive endonucleases by aqueous nanoparticle suspension of methanophosphonate fullerene derivatives and its mechanisms

Aqueous nanoparticle suspension of fullerene and its derivatives are currently attracting much attention. To determine the effects of aqueous nanoparticle suspension of a mono-methanophosphonate fullerene and bis-methanophosphonate fullerene (denoted as n-MMPF and n-BMPF, respectively) on the activities of DNA restrictive endonucleases, plasmid pEGFP-N1 was cleaved at a single but differently restrictive site by EcoR I, BamH I, and isozymes Cfr9 I and Xma I, respectively. Both n-MMPF and n-BMPF inhibited the activity of EcoR I, while n-BMPF exhibited stronger inhibition than n-MMPF. Addition of n-BMPF into reaction mixtures inhibited the activities of all the four enzymes, and IC50 values for EcoR I, BamH I, Cfr9 I and Xma I were 4.3, >30, 11.7 and 8.3 μmol/L, respectively. When EcoR I was completely inhibited by n-BMPF, addition of excess amounts of pEGFP-N1 could not produce the product linear plasmid; however, increase of EcoR I amounts antagonized EcoR I inhibition of n-BMPF. Two scavengers of reactive oxygen species (ROS), mannitol and sodium azide at the concentrations of 2-10 mmol/L, did not reverse inhibition of n-BMPF, implying that this inhibition probably is not correlated to ROS. These results suggested that aqueous nano-fullerenes might act as inhibitors of DNA restrictive endonucleases.

HUMAN CHROMOSOME BANDING WITH RESTRICTION ENDONUCLEASES HaeIII,HinfI AND PstI

Human chromosome banding was carried out with restriction endonucleases (REs)HaeⅢ, HinfⅠ, PstⅠ and the effects of some factors on it were examined. HaeⅢ induced Cand G banding patterns, HinfⅠ induced negative C bands but the centromeric heterochro-matin of chromosomes 3 and 4 remained selectively dark-stained. A new heteromorphismof C banded region was discovered in chromosome 4. PstⅠ induced G-like banding pattern.The duration of enzymatic digestion, the concentration of glycerol in the reaction buffer,as well as the ageing and heating of chromosome preparations all had a significant influ-ence on the banding effects of the REs.

Advances in homology directed genetic engineering of human pluripotent and adult stem cells

The ability to introduce precise genomic modifications in human cells has profound implications for both basic and applied research in stem cells, ranging from identification of genes regulating stem cell self-renewal and multilineage differentiation to therapeutic gene correction and creation of in vitro models of human diseases. However, the overall efficiency of this process is challenged by several factors including inefficient gene delivery into stem cells and low rates of homology directed site-specific targeting. Recent studies report the development of novel techniques to improve gene targeting efficiencies in human stem cells; these methods include molecular engineering of viral vectors to efficiently deliver episomal genetic sequences that can participate in homology directed targeting, as well as the design of synthetic proteins that can introduce double-stranded breaks in DNA to initiate such recombination events. This review focuses on the potential of these new technologies to precisely alter the human stem cell genome and also highlights the possibilities offered by the combination of these complementary strategies.

Progress and prospects of engineered sequence-specific DNA modulating technologies for the management of liver diseases

Liver diseases are one of the leading causes of mortality in the world. The hepatic illnesses, which include inherited metabolic disorders, hemophilias and viralhepatitides, are complex and currently difficult to treat. The maturation of gene therapy has heralded new avenues for developing effective intervention for these diseases. DNA modification using gene therapy is now possible and available technology may be exploited to achieve long term therapeutic benefit. The ability to edit DNA sequences specifically is of paramount importance to advance gene therapy for application to liver diseases. Recent development of technologies that allow for this has resulted in rapid advancement of gene therapy to treat several chronic illnesses. Improvements in application of derivatives of zinc finger proteins(ZFPs), transcription activator-like effectors(TALEs), homing endonucleases(HEs) and clustered regularly interspaced palindromic repeats(CRISPR) and CRISPR associated(Cas) systems have been particularly important. These sequence-specific technologies may be used to modify genes permanently and also to alter gene transcription for therapeutic purposes. This review describes progress in development of ZFPs, TALEs, HEs and CRISPR/Cas for application to treating liver diseases.

油桐尺蠖核多角体病毒的基因型变异

本文用AvaⅡ、BamHⅠ、EcoRⅠ和HindⅢ四种限制性内切酶对油桐尺蠖核多角体病毒(Buzvra suppressaria Nuclear PolyhedrosisVirus)DNA作酶解图谱分析,比较了湖北、湖南、四川三个分离株的基因组。三者图谱基本相似,但少数片段略有不同。亚克分子片段的存在表明野生的油桐尺蠖核多角体病毒有基因型变异,包含着数个基因型变种。

三种检测内源性基因修饰方法比较

为获得准确的突变信息,除直接测序外,实验初步确定了3种人工核酸酶生物学活性检测方法。利用Surveyor nuclease、T7E1(T7 Endonuclease 1)和HRM(High resolution melt),均以变性退火突变型和野生型DNA序列形成扭曲的双螺旋DNA(distorted duplex DNA)为基础的3种人工核酸酶生物学活性检测方法,确定目标位点是否发生突变。实验成功检测出作用于绵羊MNST基因第一外显子的CRISPR/Cas9和第三外显子的TALEN目标位点发生突变,并对3种检测方法的结果和特点进行了分析比较,得出3种检测方法的优缺点,为实验室分析确定细胞利用非同源末端连接修复DNA双链断裂结果提供参考。

APE1 polymorphisms are associated with colorectal cancer susceptibility in Chinese Hans

AIM:To study the association between four base excision repair gene polymorphisms and colorectal cancer risk in a Chinese population.METHODS:Two hundred forty-seven colorectal cancer(CRC)patients and three hundred cancer-free controls were enrolled in this study.Four polymorphisms(OGG1Ser326Cys,APE1 Asp148Glu,-141T/G in the promoter region,and XRCC1 Arg399Gln)in components of the base excision repair pathway were determined in patient blood samples using polymerase chain reaction with confronting two-pair primers.The baseline information included age,gender,family history of cancer,and three behavioral factors[smoking status,alcohol consumption,and body mass index(BMI)].χ2tests were used to assess the Hardy-Weinberg equilibrium,the distributions of baseline characteristics,and the four gene polymorphisms between the cases and controls.Multivariate logistic regression analyses were conducted to analyze the correlations between the four polymorphisms and CRC risk,adjusted by the baseline characteristics.Likelihood ratio tests were performed to analyze the gene-behavior interactions of smoking status,alcohol consumption,and BMI on polymorphisms and CRC susceptibility.RESULTS:The APE1 148 Glu/Glu genotype was significantly associated with an increased risk of colorectal cancer(OR=2.411,95%CI:1.497-3.886,P<0.001relative to Asp/Asp genotype).There were no associations between OGG1,XRCC1,or APE1 promoter polymorphisms and CRC risk.A multivariate analysis including three behavioral factors showed that the APE1 148Glu/Glu genotype was associated with an increased risk for CRC among both smokers and non-smokers,non-drinkers and individuals with a BMI≥25 kg/m2(ORs=2.356,3.299,2.654,and 2.581,respectively).The XRCC1 399 Arg/Gln genotype was associated with a decreased risk of CRC among smokers and drinkers(OR=0.289,95%CI:0.152-0.548,P<0.001,and OR=0.327,95%CI:0.158-0.673,P<0.05,respectively).The APE1 promoter polymorphism-141 T/G genotype was associated with a reduced risk of colorectal cancer among subjects with a BMI<25 kg/m2(OR=0.214,95%CI:0.069-0.660,P<0.05 relative to T/T genotype).There were significant gene-behavior interactions between smoking status and XRCC1 Arg399Gln,as well as BMI and APE1-141T/G polymorphism(all P<0.05).CONCLUSION:APE1 Asp148Glu is associated with increased CRC risk and smoking alters the association between XRCC1 Arg399Gln and CRC risk in the Chinese Han population.

Creation of targeted inversion mutations in plants using an RNA-guided endonuclease

Inversions are DNA rearrangements that are essential for plant gene evolution and adaptation to environmental changes. We demonstrate the creation of targeted inversions and previously reported targeted deletion mutations via delivery of a pair of RNA-guided endonucleases(RGENs) of CRISPR/Cas9. The efficiencies of the targeted inversions were2.6% and 2.2% in the Arabidopsis FLOWERING TIME(At FT) and TERMINAL FLOWER 1(At TFL1)loci, respectively. Thus, we successfully established an approach that can potentially be used to introduce targeted DNA inversions of interest for functional studies and crop improvement.

Relationship between apurinic endonuclease 1 Asp148Glu polymorphism and gastrointestinal cancer risk: An updated meta-analysis

AIM:To evaluate the relationship between apurinic endonuclease 1(APE1) Asp148 Glu polymorphism and the susceptibility to gastrointestinal(GI) cancers.METHODS:We searched Pub Med, ISI Web of Knowledge, and Chinese National Knowledge Infrastructure(CNKI) databases updated on July 15, 2014 for relevant studies.Only case-control studies comparing APE1 Asp148 Glu polymorphism and GI cancer risk were included.We excluded studies reporting only standardized incidence ratios without control groups and those without detailed genotyping data.Meta-analysis was performed on 17 studies involving 4856 cancer patients and 6136 cancer-free controls.Review Manager version 5.1 was used to perform the meta-analysis.The pooled odds ratios(ORs) and 95% confidence intervals(CIs) were estimated under the allele contrast, homozygous, heterozygous, dominant and recessive genetic models.We also conducted subgroup analyses stratified by ethnicity and cancer type.Publication bias was evaluated using Begg's test.RESULTS:The meta-analysis showed a significant association between APE1 Asp148Glu polymorphism and GI cancer risk in three genetic models in the overall population(G vs T:OR=1.18;95%CI:1.05-1.32;TG vs TT:OR=1.28;95%CI:1.08-1.52;TG+GG vs TT:OR=1.32;95%CI:1.10-1.57).Stratified analysis by ethnicity revealed a statistically increased GI cancer risk in Asians(G vs T:OR=1.27;95%CI:1.07-1.51;GG vs TT:OR=1.58;95%CI:1.05-2.38;TG vs TT:OR=1.30;95%CI,1.01-1.67;and TG+GG vs TT:OR=1.38;95%CI:1.07-1.78),but not in Caucasians.Furthersubgroup analysis by cancer type indicated that APE1Asp148Glu polymorphism may contribute to gastric cancer risk.However,Asp148Glu has no significant association with colorectal or esophageal cancer risk in any genetic model.CONCLUSION:This meta-analysis suggests that the APE1 Asp148Glu polymorphism G allele is associated with an increased GI cancer risk,especially in gastric cancer.

INACTIVATION OF THE CDKN2/pl6 GENE INDUCED BY METHYLATION AT 5'-CpG ISLAND AND ITS RELATION TO LUNG CANCER

Objectives: To investigate the methylation status of the CDKN2/pl6 gene 5′-CpG island, and study the relationship between the CDKN2/pl6 gene inactivation by methylation and lung cancer. Methods: Genomic DNA extracted from the specimens of lung cancer and normal lung tissue was digested with methylation-sensitive endonucleases, and Southern blotting was used to analyze the methylation status of the CDKN2/pl6 gene in 89 cases of lung cancer and 10 cases of normal lung tissue. Results: In 89 cases of lung cancer studied, the CDKN2/pl6 gene was shown to be methylated in 21 cases with the total methylation rate of 23.6% (21/89), in which there were 15 cases (16.9%) methylated atSmaI sites, 12 cases (13.5%) atSmaI sites and 6 cases at bothSmaI andSacII sites. The methylation of the CDKN2/pl6 gene occurred in 17 among 42 of pl6 protein negative cases of lung cancer with a rate of 40.5% (17/42), and in 3 among 47 of pl6 protein positive cases with a rate of 6.4% (3/47). The CDKN2/pl6 gene was not methylated in 10 cases of normal lung tissue. Conclusion: The aberrant methylation of the CDKN2/pl6 gene 5′-CpG island is probably an important mechanism of the gene inactivation, it may be involved in the genesis and progress of lung cancer.

Differential expression of hepatic apurinic/apyrimidinic endonuclease 1,a DNA repair enzyme,in chronic hepatitis

AIM:To determine hepatic expression of apurinic/apyrimidinic endonuclease 1(APE-1)and 8-hydroxydeoxyguanosine(8-OHdG)in patients with chronic hepatitis B and C.METHODS:Liver biopsies were obtained from 27 patients with chronic hepatitis B virus(HBV),30 with chronic hepatitis C virus(HCV),6 with autoimmune hepatitis(AIH),and 6 with primary biliary cirrhosis(PBC).Normal liver tissue was obtained from surgical resection specimens of four patients.Hepatic APE-1 protein and mRNA expression were assayed by Western blot and by real-time polymerase chain reaction,respectively.Hepatocellular APE-1 and 8-OHdG expression were determined by immunohistochemistry.RESULTS:The staining intensity of hepatocellular nuclear APE-1 was lower in the HBV group than in the other groups(P < 0.05).Hepatic APE-1 protein levels were reduced in the HBV group relative to the other groups.Hepatic APE-1 mRNA levels were also lower in the HBV group.The proportion of hepatocytes with 8-OHdG-positive nuclei was increased in the HCV,AIH and PBC groups(P < 0.05),but not in the HBV group.Hepatocellular nuclear APE-1 levels were positively correlated with hepatocellular 8-OHdG levels in both the HBV and HCV groups(HBV,r = 0.34,P < 0.05;HCV,r = 0.54,P < 0.01).CONCLUSION:An imbalance between oxidative DNA damage and APE-1 expression may contribute to hepatocarcinogenesis in chronic viral hepatitis.

Alteration of the Specificity of PstⅠ Restriction Endonuclease

The influence of factors on the subst rate-specificity of Pst Ⅰ restric tion endonuclease has been studied with the method of electrophoresis. The resu lts show that, the specificity of Pst Ⅰ almost can not be influenced by th e sin gle alteration of the concentration of Tris·HCl, Mg 2+ or Na+ in the rea ction s ystem, but it can be altered by the reduction of any two of them. The specificit y can not be altered by the single alteration of pH or the replacement of Mg 2+ w ith Mn 2+. The addition of glycerol or dimethylsulphoxide (DMSO) to the rea ction system results in the relaxation of the substrate-specificity of Pst Ⅰ , b ut dim ethylmethylformide, glycol and ethyl alcohol can not bring about the alteration of Pst Ⅰ specificity. Through the method of cloning and sequencing, the nuc leot ides of No.1 and 6 in the recognition sequence of Pst Ⅰ have changed (1C→A or 6 G→T). Used with the enzyme analysis of an artificially synthetic DNA segment co ntaining a special sequence, the nucleotides of No.1 and 6 have both changed (1C →A and 6G→T). The recognition sequence of Pst Ⅰ is speculated to be chan ged from CTGCA↓G to TGCA↓.

Kinetic Studies on the Irreversible Inhibition of Restriction Endonuclease Pst I by Site-Specific Inhibitors

The irreversible modifying effects onPst I of several inhibitors have been studied with the irreversible inhibition kinetic theory of single substrate reaction provided by Tsou, C. L. Pyridoxal phosphate (PLP), p-chloromercuribenzoic acid (PCMB), diisopropyl fluorophosphate (DEP), 2,3-diacetyl (DAC) and N-ethyl-5-phenylisoxazoliun-3′-sulfonate (woodward's reagent K, WRK) modify the lysine, cysine, serteine, arginine and carboxyl groups of the protein molecule respectively. These five inhibitors have been found to inhibit both the prime activity and star activity ofPst I. Used with the irreversible inhibition theory, the apparent inhibition rate constant,A and the microcosmic inhibition rate constants,k +0 andk′ +0 of every inhibitor were calculated. We also found that their inhibition effects belong to the noncompetitive irreversible inhibition. Results show that among the groups to be modified, some have nothing to do with the combination with the substrate, and some may have, but any of them isn't the only factor involved in the specific binding. Despite all this, they may take part in the catalysis of enzyme or have important effects on maintaining the active structure of enzyme molecules. Furthermore, serine and arginine residues are related to the alteration ofPst I conformation and then influence the ability ofPst I recognizing and incising DNA specifically.

Detection of Low-abundance Point Mutations by Competitive Strand Assisted Endonuclease Ⅳ Signal Amplification System

Genetic mutations are important molecular biomarkers for cancer diagnosis and surveillance. Therefore, the development of methods for mutation detection characterized with straightforward, highly specific and sensitive to low-level mutations within various sequence contexts is extremely needed. Although some of the currently available methods have shown very encouraging results, their discrimination efficiency is still very low. Herein, we demonstrate a fluorescent probe coupled with blocker and property of melting temperature discrimination, which is able to identify the presence of known or unknown single-base variations at abundances down to 0.1% within 20 min. The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 10.15–38.48. The method is sequence independent, which assures a wide range of application. The new method would be an ideal choice for high-throughput in vitro diagnosis and precise clinical treatment.

Sensitive detection of alkaline phosphatase based on terminal deoxynucleotidyl transferase and endonuclease Ⅳ-assisted exponential signal amplification

Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods.Terminal deoxynucleotidyl transferase(TdT)catalyzes continuous polymerization of deoxynucleotide triphosphates at the 30-OH end of single-stranded DNA in the absence of a template.In this study,we developed a highly sensitive and selective method based on TdT and endonuclease Ⅳ(Endo Ⅳ)to quantify ALP activity.After ALP hydrolyzes the 30-PO_(4) end of the substrate and generates 30-OH,TdT can effectively elongate the 30-OH end with deoxynucleotide adenine triphosphate(dATP)and produce a poly A tail,which can be detected by the poly T probes.Endo Ⅳ digests the AP site in poly T probes to generate a fluorescent signal and a new 30-OH end,leading to the generation of exponential fluorescence signal amplification.The substrate for TdT elongation was optimized,and a limit of detection of 4.3×10^(-3) U/L was achieved for ALP by the optimized substrate structure.This method can also detect ALP in the cell lysate of a single cell.This work has potential applications in disease diagnosis and biomedical detection.

Tea polyphenols increase X-ray repair cross-complementing protein 1 and apurinic/apyrimidinic endonuclease/redox factor-1 expression in the hippocampus of rats during cerebral ischemia/reperfusion injury

Recent studies have shown that tea polyphenols can cross the blood-brain barrier, inhibit apoptosis and play a neuroprotective role against cerebral ischemia. Furthermore, tea polyphenols can decrease DNA damage caused by free radicals. We hypothesized that tea polyphenols repair DNA damage and inhibit neuronal apoptosis during global cerebral ischemia/reperfusion. To test this hypothesis, we employed a rat model of global cerebral ischemia/reperfusion. We demonstrated that intraperitoneal injection of tea polyphenols immediately after reperfusion significantly reduced apoptosis in the hippocampal CA1 region; this effect started 6 hours following reperfusion. Immunohistochemical staining showed that tea polyphenols could reverse the ischemia/reperfusion-induced reduction in the expression of DNA repair proteins, X-ray repair cross-complementing protein 1 and apudnic/apyrimidinic endonuclease/redox factor-1 starting at 2 hours. Both effects lasted at least 72 hours. These experimental findings suggest that tea polyphenols promote DNA damage repair and protect against apoptosis in the brain.

APEX1 protects against oxidative damage-induced cardiomyocyte apoptosis

Apurine/pyrimidine-free endonuclease 1(APEX1)is a multifunctional enzyme that contributes to oxidization-mediated DNA-cleaved base excision repair and redox activation of transcription factors.However,the role of APEX1 during cardiomyocyte oxidative stress injury is not completely understood.In the present study,whether APEX1 protects oxidative damage-induced cardiomyocytes was investigated.mRNA and protein expression levels of APEX1 were downregulated in the mouse model of cardiac ischemia-reperfusion injury.Furthermore,the expression of APEX1 in hydrogen peroxide(H 2 O 2)-treated neonatal mice cardiomyocytes was also decreased.APEX1 knockdown aggravated H 2 O 2-treated cardiomyocyte apoptosis indexes.By contrast,APEX1 overexpression reversed H 2 O 2-induced oxidative damage,as demonstrated by decreased caspase 3 and Bax expression levels.Moreover,homeobox A5 upregulated APEX1.The results of the present study indicated that APEX1 displayed protective effects against oxidative damage,suggesting that APEX1 may serve as a unique protective strategy for cardiac ischemia-reperfusion injury.

Application of Artificially Induced Double-strand Breaks (DSB) and Triplex-forming Oligonucleotides (TFO) in the Improvement of Gene Targeting Efficiency

Gene targeting technology is an important means to investigate gene functions, but its efficiency of gene targeting is very low, especially for somatic cell targeting. Artificially induced double-strand breaks （DSB） and triplex forming oligonucleotide （TFO） are currently developed methods to improve the targeting efficiency. This paper summarized the basic principles, design ideas and application in gene targeting efficiency improvement of these two methods, analyzed and com- pared their characteristics, and finally proposed prospects for their future development.