REP-PCR分型法用于白假丝酵母菌群分析

目的运用Agilent生物分析仪对临床白假丝酵母菌进行重复序列（REP）-聚合酶链反应（PCR）分型,并分析其流行趋势。方法设计REP引物,进行PCR扩增,用DL7500 Labchip芯片在Agilent 2100生物分析仪上对产物做微流电泳,获得虚拟凝胶视图,再运用GelCompareⅡ软件对其DNA指纹图谱做聚类分析。结果白假丝酵母菌菌株指纹图谱相似度较高,〉80%以上。被分析的100株临床株可分为9个基因型。结论REP-PCR的Agilent 2100同源性分析具有快速、易于操作、高重复性和高分辨率等优点,可广泛用于流行病学调查与研究。     

Agilent2100分析仪在鲍曼不动杆菌同源性分析中的应用

目的Agilent 2100分析仪在基因组外非编码重复序列片段的聚合酶链反应(REP-PCR)同源性分析中的应用并评估该系统。方法应用REP设计引物并进行PCR扩增,用Agilent2100分析仪DL7500 Labch ip芯片进行产物分析。结果51株碳青霉烯类耐药鲍曼不动杆菌中,耐药菌株分为4大型别(A、B、C、D),其中A型共48株,分别为A1型33株,A2型13株,A3型和A4型各1株;B、C、D型各1株;18株碳青霉烯类敏感菌株中,有2株与A1型别一致,其他菌株型别都不一致。结论基于REP-PCR的Agilent2100同源性分析具有快速、易于操作、高重复性和高分辨率、结果定量处理和多种形式输出等优点,可作为临床同源性分析鉴定和分型的一线工具。     

Agilent2100分析仪在鲍曼不动杆菌同源性分析中的应用

目的Agilent2100分析仪在基因组外非编码重复序列片段的聚合酶链反应（REP-PCR）同源性分析中的应用并评估该系统。方法应用REP设计引物并进行PCR扩增,用Agilent2100分析仪DL7500 Labchip芯片进行产物分析。结果51株碳青霉烯类耐药鲍曼不动杆菌中,耐药菌株分为4大型别（A、B、C、D）,其中A型共48株,分别为A1型33株,A2型13株,A3型和A4型各1株;B、C、D型各1株;18株碳青霉烯类敏感菌株中,有2株与A1型别一致,其他菌株型别都不一致。结论基于REP-PCR的Agilent2100同源性分析具有快速、易于操作、高重复性和高分辨率、结果定量处理和多种形式输出等优点,可作为临床同源性分析鉴定和分型的一线工具。     

Detection and quantification of heteroplasmic mutant mitochondrial DNA by real-time amplification refractory mutation system quantitative PCR analysis: a single-step approach

BACKGROUND: The A3243G mitochondrial tRNA leu(UUR) point mutation causes mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, the most common mitochondrial DNA (mtDNA) disorder, and is also found in patients with maternally inherited diabetes and deafness syndrome (MIDD). To correlate disease manifestation with mutation loads, it is necessary to measure the percentage of the A3243G mtDNA mutation. METHODS: To reliably quantify low proportions of the mutant mtDNA, we developed a real-time amplification refractory mutation system quantitative PCR (ARMS-qPCR) assay. We validated the method with experimental samples containing known proportions of mutant A3243G mtDNA generated by mixing known amounts of cloned plasmid DNA containing either the wild-type or the mutant sequences. RESULTS: A correlation coefficient of 0.9995 between the expected and observed values for the proportions of mutant A3243G in the experimental samples was found. Evaluation of a total of 36 patient DNA samples demonstrated consistent results between PCR-restriction fragment length polymorphism (RFLP) analysis and real-time ARMS-qPCR. However, the latter method was much more sensitive for detecting low percentages of mutant heteroplasmy. Three samples contained allele-specific oligonucleotide-detectable but RFLP-undetectable mutations. CONCLUSIONS: The real-time ARMS-qPCR method provides rapid, reliable, one-step quantitative detection of heteroplasmic mutant mtDNA.     

Comprehensive scanning of the entire mitochondrial genome for mutations

BACKGROUND: Definitive molecular diagnosis of mitochondrial disorders has been greatly hindered by the tremendous clinical and genetic heterogeneity, the heteroplasmic condition of pathogenic mutations, and the presence of numerous homoplasmic mitochondrial DNA (mtDNA) variations with unknown significance. We used temporal temperature gradient gel electrophoresis (TTGE) to detect heteroplasmic mutations from homoplasmic variations in the whole mitochondrial genome. METHODS: We screened 179 unrelated patients by TTGE with use of 32 overlapping primer pairs. Mutations were identified by direct sequencing of the PCR products and confirmed by PCR with allele-specific oligonucleotide or restriction fragment length polymorphism analysis. RESULTS: We detected 71 heteroplasmic and 647 homoplasmic banding patterns. Sequencing of the heteroplasmic fragments identified 68 distinct novel mutations and 132 reported sequence variations and mutations; most of them occurred only once. The deleterious nature of some of the novel mutations was established by analyzing the asymptomatic family members and the biochemical and molecular characteristics of the mutation. When the number of mutations was normalized to the size of the region, the occurrence of mutations was 2.4 times more frequent in the tRNA genes than in the mRNA (protein coding) regions. CONCLUSIONS: Screening by TTGE detects low proportions of mutant mtDNA and distinguishes heteroplasmic from homoplasmic variations. Results from comprehensive molecular analysis should be followed up with clinical correlation to establish a guideline for complete mutational analysis of the entire mitochondrial genome and to facilitate the diagnosis of mitochondrial disorders.     

Rapid diagnosis of familial defective apolipoprotein B-100

A method is described for the rapid, economic and non-radioactive examination of DNA samples from hypercholesterolaemic patients for familial defective apolipoprotein B-100, using a modified polymerase chain reaction (PCR) protocol and restriction enzyme isoform genotyping. Because of the high prevalence of familial defective apolipoprotein B-100, which is estimated to be one in 500 in most screened general populations, interest is focussed on a simple method for detection of this point mutation. In our protocol a diagnostic restriction site is created by PCR, using a specifically designed partly mismatched primer. In the case of familial defective apolipoprotein B-100 the amplified DNA segment contains an additional ScaI site, whereas DNA amplified from the normal allele is resistant to ScaI digestion. A rapid differentiation between the two alleles is achieved by agarose gel electrophoresis of the ScaI-digested PCR product.     

Peroxynitrite-induced mitochondrial and endonuclease-mediated nuclear DNA damage in acetaminophen hepatotoxicity

Intracellular sources of peroxynitrite formation and potential targets for this powerful oxidant and nitrating agent have not been identified after acetaminophen (AAP) overdose. Therefore, we tested the hypothesis that peroxynitrite generated in mitochondria may be responsible for mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage. C3Heb/FeJ mice were treated with 300 mg/kg AAP and monitored for up to 12 h. Loss of mtDNA (assayed by slot blot hybridization) and substantial nDNA fragmentation (evaluated by anti-histone enzyme-linked immunosorbent assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and agarose gel electrophoresis) were observed as early as 3 h after AAP overdose. Analysis of nitrotyrosine protein adducts in subcellular fractions established that peroxynitrite was generated predominantly in mitochondria beginning at 1 h after AAP injection. Delayed treatment with a bolus dose of glutathione (GSH) accelerated the recovery of mitochondrial glutathione, which then effectively scavenged peroxynitrite. However, mtDNA loss was only partially prevented. Despite the absence of nitrotyrosine adducts in the nucleus after AAP overdose, nDNA damage was almost completely eliminated with GSH administration. A direct comparison of nDNA damage after AAP overdose with nDNA fragmentation during tumor necrosis factor receptor-mediated apoptosis showed similar DNA ladders on agarose gels but quantitatively different results in three other assays. We conclude that peroxynitrite may be partially responsible for mtDNA loss but is not directly involved in nDNA damage. In contrast, nDNA fragmentation after AAP overdose is not caused by caspase-activated DNase but most likely by other intracellular DNase(s), whose activation is dependent on the mitochondrial oxidant stress and peroxynitrite formation.     

A gel electrophoresis method for detection of mitochondrial DNA mutation (3243 tRNA(Leu (UUR))) applied to a Norwegian family with diabetes mellitus and hearing loss

Blood cells of selected patients from a large Norwegian family with maternally transmitted diabetes mellitus, hearing loss and muscular dysfunction were screened for possible A3243G mutation tRNA(Leu (UUR)) in mitochondrial DNA. We selected 7 patients from 3 of the 4 generations of the family and 10 unrelated healthy control subjects for mutation analysis using denaturing gradient gel electrophoresis (DGGE) and both manual and automated DNA sequencing. The A3243G mutation was found in peripheral blood cells of all 7 patients, but in none of the controls. The mutation was in the form of heteroplasmy and the amount of mutant DNA was found to be between 10% and 35% of total mtDNA in individual patients. This is the first report of a Norwegian family with maternally inherited diabetes and hearing loss carrying the A3243G mutation in mitochondrial DNA.     

A gel electrophoresis method for detection of mitochondrial DNA mutation (3243 tRNALeu (UUR)) applied to a Norwegian family with diabetes mellitus and hearing loss

Blood cells of selected patients from a large Norwegian family with maternally transmitted diabetes mellitus, hearing loss and muscular dysfunction were screened for possible A3243G mutation tRNA^{Leu (UUR)} in mitochondrial DNA. We selected 7 patients from 3 of the 4 generations of the family and 10 unrelated healthy control subjects for mutation analysis using denaturing gradient gel electrophoresis (DGGE) and both manual and automated DNA sequencing. The A3243G mutation was found in peripheral blood cells of all 7 patients, but in none of the controls. The mutation was in the form of heteroplasmy and the amount of mutant DNA was found to be between 10% and 35% of total mtDNA in individual patients. This is the first report of a Norwegian family with maternally inherited diabetes and hearing loss carrying the A3243G mutation in mitochondrial DNA.     

Detection of common disease-causing mutations in mitochondrial DNA (mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes MTTL1 3243 A>G and myoclonic epilepsy associated with ragged-red fibers MTTK 8344A>G) by real-time polymerase chain reaction

The 3243A>G mutation in the MTTL1 (tRNA(Leu)) gene and the 8344A>G mutation in the MTTK (tRNA(Lys)) gene are the most common mutations found in mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes and myoclonic epilepsy associated with ragged-red fibers, respectively. These mitochondrial DNA mutations are usually detected by conventional polymerase chain reaction followed by restriction enzyme digestion and gel electrophoresis. We developed a LightCycler real-time polymerase chain reaction assay to detect these two mutations based on fluorescence resonance energy transfer technology and melting curve analysis. Primers and fluorescence-labeled hybridization probes were designed so that the sensor probe spans the mutation site. The observed melting temperatures differed in the mutant and wild-type DNA by 9 degrees C for the MTTL1 gene and 6 degrees C for the MTTK gene. This method correctly identified all 10 samples that were 3243A>G mutation-positive, all 4 samples that were 8344A>G mutation-positive, and all 30 samples that were negative for both mutations, as previously identified by traditional gel-based methods. This LightCycler assay is a rapid and reliable technique for molecular diagnosis of these mitochondrial gene mutations.     

吉林省非综合征型耳聋分子病因学分析——线粒体DNA 12SrRNA1555位点突变基因筛查

目的 探讨吉林省耳聋人群的病因学特征,考察本地区非综合征型耳聋线粒体基因（mtDNA）12SrRNAA1555G突变频率。方法 收集长春市聋哑学校129例NSHI学生外周静脉血,提取mtDNA,PCR扩增目的基因片段,限制性内切酶（Alw26I）酶切分析,检测mtDNA A555G突变。结果 被检测的129例NSHI学生（AmAn耳毒性致聋者37例）,mtDNA A1555G突变阳性者3例,其中2例为初步确定为AmAn耳毒性致聋者,推测本地区NSHI耳聋mtDNAA1555G的突变频率为2.33%,由AmAn耳毒性致NSHI人群中mtDNA A1555G的突变频率为5.41%。结论 AmAn耳毒性致聋是吉林省非综合征型耳聋的重要致病原因。通过对特定人群进行mtDNA A1555G突变基因的筛查,并对阳性个体进行早期干预,可降低药物性耳聋的发病率。     

苦参碱诱导多发性骨髓瘤细胞株RPMI8226凋亡的实验研究

本研究探讨苦参碱对人骨髓瘤细胞株RPMI8226的作用及其机制。用不同浓度苦参碱处理RPMI8226细胞24、48小时,通过形态学观察、Annexin-V分析、DNA琼脂糖电泳、线粒体膜电位检测观察苦参碱对RPMI8226细胞的促凋亡作用。结果表明：RPMI8226细胞经苦参碱处理后,出现典型的细胞凋亡形态,凋亡早期细胞膜外翻,DNA琼脂糖电泳出现典型的梯状结构,线粒体膜电位崩塌。结论：苦参碱能有效地诱导骨髓瘤细胞株RPMI8226细胞凋亡,凋亡率与药物剂量和作用时间呈依赖性。     

High-throughput mitochondrial genome screening method for nonmelanoma skin cancer using multiplexed temperature gradient capillary electrophoresis

BACKGROUND: We explored the utility of multiplexed temperature gradient capillary electrophoresis (TGCE) as a screening tool for identifying genetic changes in the human mitochondrial genome. We examined changes in mitochondrial DNA (mtDNA) in nonmelanoma skin cancers (NMSCs), using TGCE to resolve genetic differences contained within the tumors compared with the control DNA. METHODS: The entire mtDNA from NMSC tissue samples was amplified in 17 overlapping amplicons averaging 1.1 kb in size. Fourteen of these amplicons were digested with restriction endonucleases into as many as five smaller analyzable fragments. Digested tumor mtDNA amplicons were annealed with digested amplicons from the control DNA to form heteroduplexes in regions of DNA mismatch. TGCE was performed in a 96-well parallel format to detect mtDNA changes in a high-throughput fashion. RESULTS: TGCE resolved heteroduplexes from homoduplexes in singlet reactions and in multiplexed assays. Using a single programmed temperature gradient, we detected 18 of 20 mtDNA changes contained within the specimens. This system was also able to detect a single nucleotide change in a fragment as large as 2 kb. CONCLUSION: Multiplexed TGCE is a sensitive and high-throughput screening tool for identifying mtDNA variations.     

Modulating mtDNA heteroplasmy by mitochondria-targeted restriction endonucleases in a 'differential multiple cleavage-site' model

The ability to manipulate mitochondrial DNA (mtDNA) heteroplasmy would provide a powerful tool to treat mitochondrial diseases. Recent studies showed that mitochondria-targeted restriction endonucleases can modify mtDNA heteroplasmy in a predictable and efficient manner if it recognizes a single site in the mutant mtDNA. However, the applicability of such model is limited to mutations that create a novel cleavage site, not present in the wild-type mtDNA. We attempted to extend this approach to a 'differential multiple cleavage site' model, where an mtDNA mutation creates an extra restriction site to the ones normally present in the wild-type mtDNA. Taking advantage of a heteroplasmic mouse model harboring two haplotypes of mtDNA (NZB/BALB) and using adenovirus as a gene vector, we delivered a mitochondria-targeted Scal restriction endonuclease to different mouse tissues. Scal recognizes five sites in the NZB mtDNA but only three in BALB mtDNA. Our results showed that changes in mtDNA heteroplasmy were obtained by the expression of mitochondria-targeted ScaI in both liver, after intravenous injection, and in skeletal muscle, after intramuscular injection. Although mtDNA depletion was an undesirable side effect, our data suggest that under a regulated expression system, mtDNA depletion could be minimized and restriction endonucleases recognizing multiple sites could have a potential for therapeutic use.     

Coenzyme Q10 deficiency associated with a mitochondrial DNA depletion syndrome: A case report

ObjectivesTo report on a case with a mitochondrial DNA (mtDNA) depletion syndrome.Design and methodsLaboratory studies were done in muscle biopsy and fibroblasts to evaluate coenzyme Q₁₀ (CoQ₁₀) status and quantify mitochondrial DNA.ResultsDecreased CoQ₁₀ values and a 78% of mtDNA depletion were detected in muscle. Mutational studies failed to reveal any pathogenic mutation in nuclear genes related with mtDNA maintenance.ConclusionsmtDNA depletion syndrome was associated with CoQ₁₀ deficiency in our patient.     

药物性耳聋相关线粒体12S rRNA突变

  目的  探讨极重度非综合征性耳聋患者线粒体DNA(mitochondrial DNA, mtDNA)1555A > G和1494C > T突变情况。  方法  选取黑龙江省佳木斯地区聋哑学校学生和北京协和医院门诊散发的极重度感音神经性耳聋患者共208例作为研究对象, 使用基因芯片方法和限制性内切酶法对其mtDNA 1555和1494两个位点进行检测, 并用直接测序的方法进行验证。  结果  208例患者中共发现1555A > G突变者10例, 该突变的携带率为4.81%;未发现1494C > T突变。  结论  mtDNA 1555A > G突变在极重度非综合征性耳聋患者中阳性率较高, 而在汉族人群中1494C > T突变较为罕见。     

Mitochondrial DNA polymorphisms of a west Algerian population (Oran region).

Mitochondrial DNA codes for enzymes involved in the cellular energetic pathway. The polymorphism of this genome has been extensively analyzed for disease associations, but can also be used to characterize anthropological distances between populations. This study presents the results of mitochondrial DNA (mtDNA) sequence variation for a population sample of 50 unrelated individuals originating from western Algeria. The samples were studied with the recently developed long PCR technique followed by RFLP analysis using six restriction endonucleases: HpaI, BamHI, HaeII, MspI, AvaII and HincII. One new morph for HpaI (named HpaI-9Alg) was detected, and was found to be derived from the combination of the already known morphs 3 and 4. mtDNA restriction endonuclease fragment patterns were analyzed for potential site gain or loss and classified into 18 mtDNA types by the sequence-comparison method. Three mtDNA types (97Alg; 2-1-7-1-1, 98Alg; 2-1-1-8-37 and 99Alg; 9Alg-1-1-1-3) were detected for the first time. Another mtDNA marker--the presence of the 9 bp deletion in the COII/tRNA(Lys) region--was also studied in the Algerian sample. No deletions were observed. Our results indicate that the Algerians are genetically related to the Israeli-Arab population, with certain characteristics found in southern Europeans and others found in sub-Saharan Africans.     

引物二聚体在目的基因克隆中的应用初探

目的 根据引物二聚体形成原理，建立一种新的克隆目的基因的方法。方法 人工合成两条或两条以上3′端互补配对的寡核苷酸链，寡核苷酸链、Taq酶和dNTP按一定比例混合，PCR扩增延伸，琼脂糖凝胶电泳鉴定结果。结果 PCR产物经电泳鉴定，可见一条清晰的特异条带。结论 此方法能合成500bp以下的短片段目的基因。     

Assessing the relative incidence of mitochondrial DNA A3243G in migraine without aura with maternal inheritance

OBJECTIVE: To determine whether patients with migraine without aura with maternal "inheritance" are affected by a monosymptomatic form of the MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes) or carry the most common mitochondrial DNA (mtDNA) mutation associated with MELAS, namely the A3243G transition in the transfer RNA (tRNA)Leu(UUR) gene. BACKGROUND: The association between migraine and abnormal mitochondrial function has been suggested on clinical, biochemical, and neuroradiological grounds. Migraine attacks with vomiting and cerebral infarctions, most often in the posterior cerebral regions, which are reminiscent of complicated migraine, are typical features of MELAS. The observation that migrainous patients have affected mothers more often than affected fathers suggests a possible role for maternally transmitted genetic factors. METHODS: We studied 25 patients with migraine with aura whose mothers were also affected. A sensitive polymerase chain reaction restriction fragment length polymorphism analysis was used to detect mutated genomes. CONCLUSIONS: We failed to detect the MELAS mutation, but migraine may still be associated with point mutations of mtDNA other than A3243G or with as-yet-unidentified nuclear DNA factors related to mitochondrial function.     

Assessing heteroplasmic load in Leber's hereditary optic neuropathy mutation 3460G->A/MT-ND1 with a real-time PCR quantitative approach

To quantify the amount of the 3460G-->A/ND1 point mutation responsible for Leber's hereditary optic neuropathy, we developed a quantitative real-time polymerase chain reaction method based on the SYBR Green assay and a new approach using the TaqMan assay. Both methods were based on the amplification refractory mutation system, comparing the heteroplasmic load quantified by restriction fragment length polymorphism in 15 Leber's hereditary optic neuropathy family members, with the results obtained using quantitative real-time polymerase chain reaction methods. The comparative evaluation of mitochondrial DNA (mtDNA) heteroplasmy from blood samples showed significant correlation between restriction fragment length polymorphism analysis, real-time SYBR Green assay, and TaqMan assay. We validated the last method by measuring experimental samples composed by a known proportion of cloned plasmids containing either the wild-type or mutant sequence, giving a correlation coefficient of 0.999 (P < 0.0001). The real-time amplification refractory mutation system polymerase chain reaction by TaqMan assay provides a rapid, reliable, sensitive, reproducible, and one-step quantitative method to detect heteroplasmic mutant mtDNA. This method allows the quantitation of a broad range of mutational load (up to 100%, down to 0.01%) on the basis of in vitro calibration, thus rendering the TaqMan assay suitable for the diagnostic analysis of heteroplasmic load in mtDNA-related disorders.