An approach to the use of stable isotopes for DNA sequencing

The sequencing of DNA by current procedures involves the use of radioisotopic or fluorescent labels. We propose that stable isotopes can be used as such labels and that the large number of stable isotopes available would allow multiplexing so that many DNA segments could be sequenced simultaneously. We have developed methods to use 57Fe2O3 to synthesize ferrocene and to attach the ferrocene to the 5' end of oligonucleotides. The 57Fe-labeled M13 universal primer functioned normally in a Sanger sequencing procedure. When a 57Fe-labeled oligonucleotide had migrated on a polyacrylamide gel it was readily located on the dried gel by scanning with resonance ionization spectroscopy (RIS) coupled with mass spectrometry. Using a 57Fe-labeled primer in a PCR reaction a 2000-bp DNA was produced that was detected by RIS on nylon membrane after agarose electrophoresis. The rapid analysis features of RIS coupled with the multispectral multiplexing possibilities of stable isotopes should significantly increase the rate of determination of DNA sequences.     

The 3′→5′ exonuclease associated with HeLa DNA polymerase epsilon

The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity.     

一种用于CYP3A5基因分型的电化学传感器阵列设计

目的：设计一种用于检测CYP3A5基因分型的电化学传感器阵列及其不同基因型的判别方法。方法：设计的电化学基体由印刷电路板（PCB）组成,该电路板包含一组金电极。每个金电极表面修饰有包含单链捕获探针的自组装单分子膜。设计中使用二茂铁做为电活性指示剂,基因分型检测是通过两种不同电势的二茂铁衍生物分别标记等位基因特异性信号探针来实现。结果：该设计能构建一种快速准确、操作简便的DNA电化学传感器阵列检测系统。结论：本文设计为使用电化学方法检测基因分型提供了一种新方法和新技术。     

一种用于CYP3A5基因分型的电化学传感器阵列设计

目的:设计一种用于检测CYP3A5基因分型的电化学传感器阵列及其不同基因型的判别方法。方法:设计的电化学基体由印刷电路板(PCB)组成,该电路板包含一组金电极。每个金电极表面修饰有包含单链捕获探针的自组装单分子膜。设计中使用二茂铁做为电活性指示剂,基因分型检测是通过两种不同电势的二茂铁衍生物分别标记等位基因特异性信号探针来实现。结果:该设计能构建一种快速准确、操作简便的DNA电化学传感器阵列检测系统。结论:本文设计为使用电化学方法检测基因分型提供了一种新方法和新技术。     

Realizing directional cloning using sticky ends produced by 3′-5′ exonuclease of Klenow fragment

The Klenow fragment (KF) has been used to make the blunt end as a tool enzyme. Its 5′-3′ polymerase activity can extend the 5′ overhanging sticky end to the blunt end, and 3′-5′ exonuclease activity can cleave the 3′ overhanging sticky end to the blunt end. The blunt end is useful for cloning. Here, we for the first time determined that a sticky end can be made by using the 3′-5′ exonuclease activity of KF. We found that KF can cleave the blunt end into certain sticky ends under controlled conditions. We optimized enzyme cleavage conditions, and characterized the cleaved sticky ends to be mainly 2 nt 5′ overhang. By using these sticky ends, we realized ligation reaction in vitro, and accomplished cloning short oligonucleotides directionally with high cloning efficiency. In some cases, this method can provide sticky end fragments in large scale for subsequent convenient cloning at low cost.     

An Extracellullar Nuclease from Bacillus Firmus VKPACU-1: Specificity and Mode of Action

An extracellular nuclease from Bacillus firmus VKPACU-1 was multifunctional enzyme, this nuclease hydrolyzed poly U rapidly and more preferentially than the other homopolyribonucleotides. Hydrolysis of RNA this enzyme released mononucleotides in the order 5′UMP > 5′AMP > 5′GMP where as in hydrolysis of DNA the mononucleotides in the order of 5′dAMP > 5′dGMP > 5′dTMP and oligonucleotides. Uridylic linkages in RNA and adenylic linkages in DNA were preferentially cleaved by the nuclease. Nuclease produced oligonucleotides having only 3’ hydroxyl and 5’ phosphate termini. Present nuclease hydrolyzed RNA and DNA released oligonucleotides as major end products and mononucleotides, suggesting an endo mode of action.     

Effect of Oligomer Length and Base Substitutions on the Cytotoxic Activity and Specific Nuclear Protein Recognition of GTn Oligonucleotides in the Human Leukemic CCRF-CEM Cell Line

Abstract

We have identified phosphodiester oligonucleotides composed of G and T bases, named GTn, which are able to inhibit the cellular growth of human cancer cell lines by recognising specific nuclear proteins. We demonstrated that GTn oligonucleotides require a length of at least 20 nucleotides in order to exert a significant cytotoxic effect and to retain the specific protein binding ability. In addition, we found that GTn cytotoxicity was lost when A or C bases were introduced at either 3′ and 5′end or within the GTn sequences.     

Ferrocene-functionalized SWCNT for electrochemical detection of T4 polynucleotide kinase activity

A novel electrochemical strategy for monitoring the activity and inhibition of T4 polynucleotide kinase (PNK) is developed by use of titanium ion (Ti(4+)) mediated signal transition coupled with signal amplification of single wall carbon nanotubes (SWCNTs). In this method, a DNA containing 5'-hydroxyl group is self-assembled onto the gold electrode and used as substrate for PNK. The biofunctionalized SWCNTs with anchor DNA and ferrocene are chosen as the signal indicator by virtue of the intrinsic 5'-phosphate end of anchor DNA and the high loading of ferrocene for electrochemical signal generation and amplification. The 5'-hydroxyl group of the substrate DNA on the electrode is phosphorylated by T4 PNK in the presence of ATP, and the resulting 5'-phosphoryl end product can be linked with the signal indicator by Ti(4+). The redox ferrocene group on the SWCNTs is grafted to the electrode and generates the electrochemical signal, the intensity of which is proportional to the activity of T4 PNK. This assay can measure activity of T4 PNK down to 0.01 UmL(-1). The developed method is a potentially useful tool in researching the interactions between proteins and nucleic acids and provides a diversified platform for a kinase activity assay.     

Multifunctional Fe₃O₄ core/Ni-Al layered double hydroxides shell nanospheres as labels for ultrasensitive electrochemical immunoassay of subgroup J of avian leukosis virus

A novel electrochemical immunosensor for ultrasensitive detection of subgroup J of avian leukosis virus (ALVs-J) was designed by using graphene sheets (GS)-layered double hydroxides (LDHs) composites modified electrode with multifunctional Fe(3)O(4) core/Ni-Al LDHs shell (LDHs@Fe(3)O(4)) nanospheres as labels. At first, the GS-LDHs were used for the immunosensor platform for improving the electronic transmission rate as well as increasing the surface area to capture a large amount of primary antibodies (Ab(1)). After that, ferrocene (Fc), secondary antibodies (Ab(2)) and horseradish peroxidase (HRP) multifunctional LDHs@Fe(3)O(4) nanospheres were used as labels with high load amount and good biological activity. Subsequently, in presence of H(2)O(2), amplified signals were obtained by an electrochemical sandwich immunoassay protocol. To embody the signal amplification property of the protocol, the analytical properties of various immunosensor platform and labels were compared in detail. Under optimal conditions, the reduction peak currents of the electrochemical immunosensor were proportional to the ALVs-J concentration over the range from 10(2.32) to 10(5.50) TCID(50)/mL with a low detection limit (180 TCID(50)/mL, S/N=3). The resulting immunosensor also displayed a good selectivity, reproducibility and stability.     

Preparation of ferrocene-functionalized gold nanoparticles by primer extension reaction on the particle surface

DNA molecules possessing multiple ferrocene (Fc) molecules as a redox active probe were prepared by the primer extension (PEX) reaction using a 2′-deoxyuridine-5′-triphosphate derivative in which Fc was connected to the C5-position of the uridine by a diethylene glycol linker. Gold nanoparticles (AuNP) covered with DNA possessing the Fc molecules were prepared by the PEX reaction on the surface. The AuNP–FcDNA conjugates exhibit a detectable electrochemical signal due to the Fc molecules. Possible application of the PEX reaction on AuNP is demonstrated for the detection of a single nucleotide mutation in the target DNA.     

MULTIPLE OLIGONUCLEOTIDE SYNTHESIS IN TANDEM ON SOLID-PHASE SUPPORTS FOR SMALL AND LARGE SCALE SYNTHESIS

Multiple oligonucleotides linked end-to-end in tandem can be synthesized by adding a nucleoside to the 5′-OH end of a prior sequence. Nucleosides with 3′-succinyl or Q-Linker arms are coupled with HBTU/DMAP. Alternatively, new phosphoramidite reagents with 3′-ester linkages can be used. Hydroxyl or amino supports can also be used as universal starting materials. Treatment with NH₄OH cleaves the 3′-ester to yield only 3′-OH groups and no unwanted 3′-phosphorylated products occur.     

Synthesis and characterization of two novel [Ru(bpy)2(phen)]2+-based electrochemiluminescent labels

Two novel electrochemiluminescent labels, bis(2,2′-bipyridine)[5-(3-carboxylic acid-propionamido)-1,10-phenanthroline]ruthenium(II) hexafluorophosphate dihydrate and bis(2,2′-bipyridine)[5-(4-carboxylic acid-butanamido)-1,10-phenanthroline]ruthenium(II) hexafluorophosphate dihydrate, were synthesized and confirmed by IRelemental analysis, and ¹H-NMR spectra were completely assigned using the ¹H-¹H COSY technique. Cyclic voltammograms with different scan rates showed quasi-reversible electrochemical behaviour of the two Ru (II) complex labels in MeCN solution. Electronic absorption, photoluminescence and electrochemiluminescence of Ru(II) complexes were also characterized. Copyright © 2000 John Wiley & Sons, Ltd.     

化学修饰对反义寡核苷酸稳定性及抗流感病毒活性的影响

为了探讨 Ａ Ｓ Ｏ Ｄ Ｎ 化学修饰形式与 Ａ Ｓ Ｏ Ｄ Ｎ 稳定性,体外细胞毒性以及抗流感病毒活性之间的关系,合成了 ７ 种不同化学修饰形式的 Ａ Ｓ Ｏ Ｄ Ｎ：硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端分别磷酸化和胆固醇修饰;３′与 ５′端硫代,中间为天然结构的混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ;天然结构 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端分别磷酸化和胆固醇修饰等．测定了 ７ 种修饰体在小鼠血清, Ｍ Ｄ Ｃ Ｋ 细胞裂解液,含 ２％ 胎牛血清的 Ｄ Ｍ Ｅ Ｍ培养液以及水中的稳定性,体外细胞毒性和在细胞水平抗流感病毒活性．结果表明,混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ,硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆固醇的修饰形式在小鼠血清, Ｍ Ｄ Ｃ Ｋ 细胞裂解液与含２％ 胎牛血清的 Ｄ Ｍ Ｅ Ｍ 培养液中稳定性相对较高,作用 ２４～４８ ｈ 仅混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ 与硫代 Ａ Ｓ Ｏ Ｄ Ｎ 发生部分降解;天然结构 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆固醇修饰体在 ２４ ｈ 内大部分降解．所有 Ａ Ｓ Ｏ Ｄ Ｎ 修饰体在水中具有很高稳定性,４８ ｈ 内未见降解作用．７ 种 Ａ Ｓ Ｏ Ｄ Ｎ 修饰形式在 Ｍ Ｄ Ｃ Ｋ 细胞中未表现明显的细胞毒性．硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆     

Interactions of Q beta replicase with Q beta RNA

The interactions of Qβ replicase with Qβ RNA were investigated by treating replicase-Qβ RNA complexes under various conditions with ribonuclease T₁, and by characterizing enzyme-bound RNA fragments recovered by a filter binding technique. Evidence for replicase binding at two internal regions of Qβ RNA was obtained. One region (at about 1250 to 1350 nucleotides from the 5′ end) overlaps with the initiation site for coat protein synthesis; this interaction is thought to be inessential for template activity but rather to be involved in the regulation of protein synthesis. Binding to this site (called the S-site) requires moderate concentrations of salt but no magnesium ions. The other region (at about 2550 to 2870 nucleotides from the 5′ end) is probably essential for template activity; binding to this site (called the M-site) is dependent on the presence of magnesium ions. The nucleotide sequences of the RNA fragments from the two sites were determined and found to have no common features. Under the conditions tested, replicase binding at the 3′ end of Qβ RNA could not be demonstrated, except when initiation of RNA synthesis was allowed to occur in the presence of GTP and host factor. If instead of intact Qβ RNA, a complete RNAase T₁ digest of Qβ RNA was allowed to bind to replicase, oligonucleotides from the S-site and the M-site, and oligonucleotides from a region close to the 3′ end, were found to have the highest affinity to the enzyme.The RNA fragments recovered in highest yield, M-2 and S-3 from the M and S-site, respectively, were isolated on a preparative scale and their enzyme binding properties were studied. In competition assays with random RNA fragments of the same size, selective binding was observed both for the M and the S-site fragment. Partial competition for replicase binding was found if M-2 and S-3 were presented simultaneously to the enzyme. Either fragment, if preincubated with replicase, caused a specific inhibition of initiation of Qβ RNA-directed RNA synthesis, without inhibiting the poly(rC)-directed reaction.The results are discussed in terms of a model of replicase-Qβ RNA recognition. Template specificity is attributed to binding of internal RNA regions to replicase, resulting in a specific spatial orientation of the RNA by which the inherently weak, but essential, interaction at the 3′ end is allowed to occur and to lead to the initiation of RNA synthesis.     

Solid Phase Synthesis of 2′, 5′-Oligoadenylates Containing 3′-Fluorinated Ribose

Abstract

2′, 5′-phosphodiester bond-linked oligoadenylate trimers with 3′-fluoro-3′-deoxyadenosine residues incorporated at specific positions of the nucleotide sequence were synthesized by the solid phase phosphite triester (phosphoramidite) method. The syntheses were in the 2′ to 5′ direction and were performed manually using commercially available microcolumns. The oligonucleotides were 5′-end phosphorylated on the support before deprotection.     

Characterization of the ribosome-protected regions of 125I-labelled rabbit globin messenger RNA

The fragments of ¹²⁵I-labelled rabbit globin messenger RNA protected from pancreatic RNAase by initiating 40 S subunits and 80 S ribosomes were analysed using the techniques of RNA sequencing. The fragments were cleaved specifically at cytidine residues generating oligonucleotides labelled in their 3′ terminal residue. Analysis of the partial digestion products of these oligonucleotides after treatment with pancreatic, T₁, U₂ and T₂ RNAase enabled their sequences to be deduced. Sequences were determined from knowledge of the specificities of the ribonucleases and then confirmed in a separate analysis making use of the known electrophoretic mobilities of each base. This combination of methods served to establish that the 40 S- and 80 S-protected fragments are related, and that both contain the initiation codon of the mRNA. The 80 S-protected fragment is about 40 bases in length whilst the 40 S-protected fragments range from 50 to more than 60 bases in length. The most prominent of these 40 S-protected fragments is about 50 bases in length and extends more towards the 5′ end of the mRNA than does the 80 S-protected fragment. It follows that 80 S ribosomes do not protect the 5′ end of the mRNA from nuclease digestion and that the 5′ terminus of rabbit globin mRNA must be at least 15 to 30 bases from the initiation codon.     

Quantitative Parametrs of Cooperative Interactions of the Oligodeoxyribonucleotides on the Complementary Template

Abstract

The cooperative interactions of oligonucleotides on the complementary template were studied using the quantitative analysis of the template alkylation with the oligonucleotides bearing covalently attached 4-[N-(2-chloroethyl)-N-methylamino]benzyl group at 5′-end. The influence of the mismatched nucleotides and the stabilizing N-(2-hydroxyethyl)phenazinium group at the 5′- and 3′-ends of the oligonucleotides on the parameters of cooperativity was evaluated.     

Affinity capture of specific DNA fragments with short synthetic sequences

The ability of short peptide nucleic acid (PNA) oligomers and oligonucleotides containing modified residues of 5-methylcitidine, 2-aminoadenosine, and 5-propynyl-2′-deoxyuridine (strong binding oligonucleotides, SBO) to affinity capture the target double-stranded DNA fragment from mixture by means of the end invasion was compared. Both types of probes were highly effective at the conditions used. The SBO-based probes may represent a handy and easily prepared alternative to PNA for selection of target DNA fragments in mixtures.