焦磷酸测序中PCR引物与测序引物的设计

利用焦磷酸测序技术进行核酸序列测定时,测序信号过低或非特异性信号过高均会导致测序失败。因此,PCR引物与测序引物的设计十分重要。本研究以rs8175347为例,通过设计具有不同多聚酶亲和指数(PPI)的PCR引物,并运用焦磷酸测序测定其扩增产物,考察了PPI值对扩增效率以及测序信号的影响;以rs914232、rs671位点为例,对比不同测序方向以及dNTP的推注顺序,考察了两者对测序结果的影响。结果表明,提高PCR引物的PPI值有助于增强扩增效率与测序结果的信号峰强度,测序方向和dNTP推注顺序的不合理选择会严重干扰部分SNP测序结果的判断。因此,在设计PCR引物时,应将多聚酶亲和指数理论与传统基于解链温度值的引物设计思路相结合,为焦磷酸测序提供高质量的测序模板;在进行定量测序时,还要综合考虑待测位点(SNP)两侧的序列,选择合适的测序方向以及dNTP的推注顺序,使测序结果更加准确。     

碱基序列标记法结合焦磷酸测序测定不同来源基因表达量

建立了一种将序列标记反转录聚合酶链反应(PCR)与焦磷酸测序技术结合的相对基因表达量测定法(简称"SRPP").先用来源特异性引物对不同来源的同一基因通过反转录标记上特异性标签,PCR后用焦磷酸测序法对扩增产物进行序列解码,使得测序结果中的序列代表基因的来源,峰高代表基因在不同来源中的相对表达量.用实时荧光定量PCR法对本方法的准确性进行了验证,结果表明,SRPP可以同时准确测定同一基因在3个不同来源中的表达量,并实际测定了Egr1基因在糖尿病、肥胖和正常小鼠肝中的表达量差异.     

人类白细胞抗原基因HLA-B*58∶01基因型的快速低成本鉴别方法

采用焦磷酸测序技术和改进的全血基因组提取方法,建立了位于6号染色体的银屑病易感基因1候选基因1(Psoriasis susceptibility 1 candidate 1,PSORS1C1)rs9263726位点的焦磷酸测序方法,检出限达到0.4 ng/μL基因组DNA,20例标本的焦磷酸法和Sanger法测序结果完全一致.利用本方法对683例标本的rs9263726位点进行测序,得到中国人群该位点野生型(GG型)分布频率为87.6％,突变的GA型分布频率为11.7％、AA型分布频率为0.7％.通过对随机选取的46例标本rs9263726位点与人类白细胞抗原基因HLA-B* 58∶01基因型的连锁关系进行分析,结果表明,该位点预测HLA-B* 58∶01基因型的灵敏度为100％、特异性为91.3％.本方法可用于HLA-B* 58∶01基因型的检测.     

污染土壤中主要石油降解基因AlkB和Nah定量检测方法的建立和应用

建立了SYBR Green I实时荧光定量聚合酶链式反应(Real Time-qPCR)检测油田污染土壤中烷烃降解基因AlkB和萘降解基因Nah的方法。比对相关降解石油菌株的GenBank序列,设计合成针对烷烃和萘降解基因扩增引物AlkBf/AlkBr和Nahf/Nahr。将纯化的常规PCR胶回收产物与pEASY-T1载体连接,转化到感受态细胞培养。提取并梯度稀释阳性克隆质粒,构建Real Time-qPCR标准测定曲线。25μL扩增体系最佳反应条件:前后引物终浓度为0.2μmol/L,12.5μL 2×TransStart Top Green qPCR SuperMix,AlkB和Nah基因最适退火温度分别为50℃和57℃。Real Time-qPCR技术显示出很高的灵敏性和重复性,比传统PCR技术灵敏度高100倍。对采集于某油田3个功能区的14土壤样品中AlkB定量检测显示,石油污染严重的采油区含有最高的AlkB拷贝数,污染较轻的生活区AlkB拷贝数最少;Nah基因分布均匀。     

牛奶α-乳白蛋白基因实时荧光定量PCR检测方法的建立

采用Taqman探针技术,建立食品中牛奶过敏原α-乳白蛋白基因的实时荧光定量PCR检测方法。根据α-乳白蛋白基因序列设计特异性引物及Taqman探针进行PCR扩增,构建质粒,经酶切鉴定测序后,建立拷贝数(copies)-Ct标准曲线。成功克隆α-乳白蛋白目的基因,建立的标准曲线在1.12×103~1.12×108 copies范围内线性关系良好,灵敏度高,液体样品检测限达到1 000copies/mL,特异性强,稳定性好。该法可用于实际商品中牛奶过敏原α-乳白蛋白组分的定量检测。     

全血改进线性指数聚合酶链式反应用于焦磷酸测序检测基因多态性

焦磷酸测序是目前基因多态性检测的主要方法之一,但是其前期的样本制备工作较为繁琐,限制了其在临床检测中的应用。为了简化焦磷酸测序的流程,本研究根据不对称PCR原理,改进了线性指数聚合酶链式反应(LATE-PCR)的引物设计方法,增加过量引物的长度和浓度,并结合全血直接扩增技术,建立了基于普通r Taq聚合酶和高p H缓冲液(Hp H Buffer)的全血改进LATE-PCR(Improved LATE-PCR,im LATE-PCR)方法。考察了方法的最优扩增体系、血液抗凝剂对其影响以及全血模板量。采用单管、一步法直接扩增出单链测序模板,成功地对24例临床血样的乙醇脱氢酶基因多态性进行了检测,检测结果可用于指导临床个体化用药。24例样本的基因型分别为ADH1B位点AA纯合6例、AG杂合14例、GG纯合4例;ADH1C位点GG纯合20例、AG杂合4例、AA纯合0例。     

我国建立未知细菌快速筛查技术

近13完成的一项“十五”国家科技攻关课题，研究人员成功建立了未知细菌快速筛查技术体系，他们以16SrRNA序列分析为基础，采取毒力和毒素基因鉴定方法，能够直接使用临床标本，在最短的时间内鉴定未知或不可培养的细菌，对细菌性新发传染病做出准确判断。科研人员建立了相关的数据库和临床标本细菌检测体系，建立了细菌毒力岛和毒素基因检测芯片，以及重要致病细菌毒素和毒力岛基因的二级核酸数据库及分析软件。其中细菌16SrDNA保守核酸序列数据库属国际首创，细菌毒力岛和毒素基因检测芯片属国内首创。该技术是将临床标本处理后，使用细菌16SrRNA的通用引物，直接进行PCR扩增、克隆和测序。然后将获得的16SrRNA序列，与已经发表的序列进行比较，同时进行毒力和毒素基因的检测和筛查，     

一种基于磁性颗粒和通用连接子扩增技术的单核苷酸多态性分型方法

提出了一种应用磁性颗粒和通用连接子扩增技术(Linker-PCR)的多位点单核苷酸多态性(SNP)分型方法. 该方法首先通过酶切将样本基因组DNA打断, 然后将通用连接子通过T4 DNA连接酶与各个酶切片段连接, 利用生物素标记的通用引物将样本进行全基因组扩增. 扩增后, 将生物素标记的Linker-PCR扩增产物固定到亲合素修饰的磁性颗粒表面, 通过与双色荧光标记的等位基因特异性探针杂交, 对待测位点进行分型. 利用该方法, 我们对10个样本MTHFR基因上的2个SNP位点进行了分型, 分型结果准确、正错配信号比大于3. 由于利用Linker-PCR技术来实现对靶序列的全基因组扩增, 该方法非常适用于大量样本的多基因多位点的SNP分型研究.     

全血直接扩增结合焦磷酸测序法测定亚甲基四氢叶酸还原酶基因多态性

亚甲基四氢叶酸还原酶基因677C>T和1298A>C两个位点的多态性与临床常用抗肿瘤药物甲氨喋呤及氟尿嘧啶的作用密切相关,对这两个位点多态性的检测能指导临床合理用药。为进一步缩短检测时间,降低检测成本,本研究建立了基于全血直接PCR的焦测序检测方法,采用"HpH Buffer"直接扩增全血模板,仅需1μL全血样本即可对两个位点进行高效扩增。扩增产物经碱变性法制备单链模板后进行焦磷酸测序,经过条件优化,仅需5μL扩增产物和1μL微球即可完成高灵敏的焦测序反应。为验证方法的准确性,检测了12例临床样本,均能正确检测两个位点的基因多态性。本研究为临床基因多态性检测提供了一种操作简便,耗时短,成本低,准确度高的方法,本方法可用于指导甲氨喋呤和5-氟尿嘧啶的个体化用药。     

一种基于磁性纳米粒子PCR的高通量SNP分型方法

利用磁性纳米粒子PCR扩增(MNPs-PCR)和等位基因特异性双色荧光探针(Cy3, Cy5)杂交, 建立了一种单核苷酸多态性(SNP)分型的新方法. 应用该方法对9个样本MTHFR基因的C677T多态进行检测, 野生和突变型样本正错配信号比大于9.0, 杂合型正错配信号比接近1.0, 分型结果经测序验证. 此方法无须产物纯化、浓缩, 扫描分型结果快速、直观, 是一种操作简单、快速、高通量、高灵敏度的分型方法.     

二氧化碳和丙烯直接合成甲基丙烯酸的NiPMo/TiO2催化剂的研究

用溶胶-凝胶法以磷钼酸(MPA)的镍盐溶液水解钛酸四丁酯制备了NiPMo/TiO2催化剂.使用ICP、 XRD、 TG-DTA、 IR、 TPD-MS和微反应技术研究了催化剂的化学组成、热稳定性、化学吸附性质和催化反应性能.杂多钼酸盐与TiO2通过O2-在TiO2表面发生了键合.在623 K下,杂多阴离子仍保持原有的Keggin结构.CO2在Lewis酸位Ni(Ⅱ)和Lewis碱位Ni-O-Mo的桥氧协同作用下生成CO2卧式吸附态Ni(Ⅱ)←O-(CO)←(O--Ni).丙烯有多种吸附态在催化剂上吸附.在563 K、 1 MPa和空速1500 h-1的反应条件下,丙烯的摩尔转化率为3.2%,产物MAA选择性为95%.     

Synthesis of 1-benzyloxypyrazin-2(1H)-one derivatives

Different approaches for the synthesis of 1-benzyloxypyrazin-2(1H)-one derivatives from simple amino acids have been investigated. A library of 33 precursors for the preparation of N-hydroxy pyrazinones was obtained in moderate to good yields.     

Toward new camptothecins. Part 6: Synthesis of crucial ketones and their use in Friedländer reaction

In the context of the preparation of camptothecin and luotonin A analogs, the synthesis of some key keto-precursors and their use in Friedländer condensation are described. This paper also focuses on the stability of these keto intermediates and emphasizes the major differences between indolizinones and pyrroloquinazolinones series. Noteworthy is also the report of some original structures isolated as by-products of some experiments.     

CoFe2O4自形成磁性液体场致结构化对磁化的影响

The Langevin paramagnetic theory can’t describe the relation between magnetization of ferrofluids and applied magnetic field. The structuralization of ferrofluids, which is considered the main influence factor of the magnetization, is regarded. The part of magnetization works is deposited when the structure is forming. This action influences the magnetization of ferrofluids directly or indirectly. On the base of the “compressing” model, the Langevin function that usually describes the magnetization of ferrofluid is modified, and a well-fitted curve is obtained. An equation of the relation between the equivalent volume fraction after being “compressed” and the intensity of magnetic field is discovered, which approximately describes the process of magnetization. The relation between the approximate initial susceptibility and the volume fraction can be obtained from modified formula.     

Highly regioselective Buchwald–Hartwig amination at C-2 of 2,4-dichloropyridine enabling a novel approach to 2,4-bisanilinopyridine (BAPyd) libraries

The highly regioselective Buchwald–Hartwig amination at C-2 of the cheap and readily accessible reagent, 2,4-dichloropyridine with a range of anilines and heterocyclic amines is described. This new methodology is robust and provides a facile access to 4-chloro-N-phenylpyridin-2-amines on 0.25 mol scale. These intermediates undergo a further Buchwald–Hartwig amination at higher temperature to enable rapid exploration of the chemical space at C-4 and to provide a library of 2,4-bisaminopyridines.     

KMnO4-mediated oxidative CN bond cleavage of tertiary amines: Synthesis of amides and sulfonamides

KMnO₄-mediated oxidative CN bond cleavage of tertiary amines producing secondary amine was introduced, which was trapped by electrophiles (acyl chloride and sulfonyl chloride) to form amides and sulfonamides. The reaction could take place at mild condition, tolerating a wide range of function groups and affording products in moderate to excellent yields.     

Chemistry of heterocyclic compounds at the A. E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences, over 50 years (Review)

The review contains a concise historical account and information on the most significant researches undertaken by the staff at the A. E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences on the Chemistry of Heterocyclic Compounds. Dedicated to Academician of the Russian Academy of Sciences B. A. Trofimov on his 70th jubilee. Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 10, pp. 1443–1502, October, 2008.     

Sulfur-directed metal-free and regiospecific methyl C(sp3)–H imidation of thioanisoles

Regiospecific and direct imidation of the methyl C(sp³)–H bond of thioanisoles is realized under mild and metal-free conditions with N-fluorobis(benzenesulfonyl)imide as an oxidant and nitrogen source. Proposed mechanism suggests that thionium ion intermediates and a Pummerer-type reaction are involved. The imidation has advantages such as high step-economy, excellent functionality tolerance, and regiospecificity, giving structurally diverse imidation products.     

A general and convenient synthesis of 4-(tosylmethyl)semicarbazones and their use in amidoalkylation of hydrogen,heteroatom, and carbon nucleophiles

A general synthesis of previously unknown semicarbazone-based α-amidoalkylating reagents, 4-(tosylmethyl)semicarbazones, has been developed. The synthesis involved three-component condensation of semicarbazones of aliphatic or aromatic aldehydes with the same or other aldehydes and p-toluenesulfinic acid. The scope and limitations of this reaction were investigated. The compounds obtained were demonstrated to be an efficient α-(4-semicarbazono)alkylating agents. They were reacted with H- (sodium borohydride), O- (sodium methylate), S- (sodium phenylthiolate), N- (pyrrolidine, sodium succinimide), P- (trialkyl phosphites), and C-nucleophiles (sodium diethyl malonate) to give the corresponding products of the tosyl group substitution, 4-substituted semicarbazones, including analogues of nitrofurazone. Among the prepared compounds tested in vitro for antibacterial and antifungal activity, three nitrofuryl-containing semicarbazones exhibited high biological activities with minimum inhibitory concentration (MIC) values of 8–32 μg/mL.     

Synthesis of novel chiral bidentate hydroxyalkyl-N-heterocyclic carbene ligands and their application in palladium-catalyzed Mizoroki–Heck couplings and asymmetric addition of diethylzinc to benzaldehyde

A small library of new chiral bidentate hydroxyalkyl-imidazolium salts 1 is conveniently synthesized on multi-gram scale from inexpensive and commercially available chiral pool amino acids. The corresponding carbenes, generated by deprotonation of imidazolium salts 1, in combination with palladium(II) chloride were tested in the Mizoroki–Heck coupling reaction. The most significant results in terms of yields and reactivities were achieved with low catalyst loading. The catalytic activities of these imidazolium salts were also investigated in the asymmetric addition of diethylzinc to benzaldehyde. The use of MgO nanoparticles as an additive in conjunction with these ligands played a crucial role in increasing the efficiency of these reactions.