Solid-phase methods for sequencing nucleic acids. III. Simultaneous sequencing of different long RNA fragments using DE 81 anion-exchange paper

A solid-phase method for simultaneous sequencing of different long RNA fragments has been developed using Whatman DE 81 anion-exchange paper as the support. The approach involves 8 operations including: immobilization of heat-denatured 3'-end labeled RNA fragment on DE 81 paper; washing; modification reactions; washing; aniline reaction; washing; RNA desorption by salt and ethanol precipitation. For modifying the RNA, the following reactions were selected for the routine: G with dimethylsulfate at 90 degrees C/sodium borohydride at 0 degrees C, A + G with diethylpyrocarbonate at 90 degrees C, U + C with hydrazine at 0 degrees C and C with hydrazine/5M NaCl at 0 degrees C. The losses of RNA material during the reactions with large excess of reactants were 50% during the reduction with NaBH4 and 30% during C-reaction. Almost no losses were observed during aniline reaction. The RNA could be recovered by desorption with 2M NaClO4 in 50-70% yield. The whole solid-phase procedure up to the sequencing gel takes about 2 hours and is much faster and more convenient than chemical RNA sequencing in solution according to Peattie, especially if many fragments are to be processed.     

An improved direct RNA sequence method; its application to Vicia faba 5.8S ribosomal RNA.

We have developed a direct read-off sequencing procedure, based on the method of Stanley and Vassilenko using E. coli 5S ribosomal RNA as a model compound. Radioactive bands were transferred from an acrylamide gel fractionation in the first dimension onto a DEAE-cellulose thin layer plate. After in situ enzymatic digestion with RNase T2, mononucleoside 3',5'-diphosphates were separated in the second dimension by electrophoresis at pH 2.3. Using this two-dimensional procedure the entire sequence of 163 residues of the previously unknown Vicia faba (broad bean) 5.8S ribosomal RNA was deduced.     

Characterization of an α-amanitin insensitive RNA polymerase from cauliflower

α-Amanitin insensitive RNA polymerase (polymerase I isolated from apical parts of the cauliflower inflorescence was highly stable for several months at − 18°. The DEAE-cellulose fraction was more effective in utilizing denatured DNA than native DNA as a template. Optimum pH for RNA synthesis was ca 7 in the reaction mixture with Tris-HCl or with Tris-maleate buffer. From the properties examined, it seems that DNA-dependent RNA polymerase I of cauliflower differs from other eucaryotic RNA polymerases.     

Simultaneous sequencing of RNA by solid-phase chemical degradation using DE 81 anion-exchange paper

A solid-phase chemical degradation method for simultaneous sequencing of RNA and RNA fragments has been developed using Whatman DE 81 anion-exchange paper as the support. The approach involves the following operations: (1) immobilization of the 3′-end labeled RNAs or RNA fragments on DE 81 paper; (2) washing; (3) modification reactions; (4) washing; (5) sorting of the paper segments; (6) aniline reaction; (7) lyophilization; (8) desorption of the RNA by TEAB; and (9) lyophilization.     

Sequence of 6S RNA of <Emphasis Type="Italic">E. coli</Emphasis>

THE principal technical difficulty in sequencing uniformly ³²P-labelled RNA is the fractionation of the complicated mixture of products present in partial enzyme digests. We have recently improved the methods for the separation of oligonucleotides larger than decanucleotides by using thin-layer chromatography on DEAE-cellulose in conjunction with ionophoresis on cellulose acetate at pH 3.5 (ref. 1). We have used this technique to establish the nucleotide sequence of the 6S RNA of Escherichia coli.     

A solid phase method of determining DNA sequence

A solid-phase method for DNA sequencing has been developed which involves immobilization of the terminally labeled DNA fragment on the DEAE-paper followed by chemical modification and cleavage at G, A + G, C + T, and C sites. As compared to the Maxam and Gilbert method, the new technique is more rapid and less laborious, being of the same efficiency.     

Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA

A solid-phase approach is described for manual and automated sequencing of plasmid DNA obtained directly from bacterial colonies through the polymerase chain reaction. The DNA fragment is selectively immobilized to magnetic beads and after strand-specific elution, the eluted strand, as well as the remaining immobilized strand, is used for bidirectional dideoxy sequencing. The solid-phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The approach is exemplified by fluorescent sequencing of a cloned Streptomyces curacoi gene having a G + C content of more than 70%.     

Purification and characterization of a GTP-pyrophosphate exchange activity from vaccinia virions. Association of the GTP-pyrophosphate exchange activity with vaccinia mRNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex (capping enzyme)

A core-associated enzyme, which catalyzes a nucleotide-pyrophosphate exchange with GTP, has been purified from vaccinia virions. The enzyme requires MgCl2 for activity, has an alkaline pH optimum, and specifically utilizes GTP as the exchanging nucleotide. The enzyme does not catalyze exchange of GMP with GTP. The GTP-PPi exchange enzyme co-purifies with vaccinia capping enzyme (RNA guanylyltransferase and RNA (guanine-7-)methyltransferase) through successive chromatography steps on DEAE-cellulose, DNA-cellulose, and phosphocellulose. GTP-PPi exchange and capping activities remain physically associated during sedimentation in a glycerol gradient. Under high salt conditions (1 M NaCl), GTP-PPi exchange, capping, and methylating activities co-sediment with an RNA triphosphatase activity and a nucleoside triphosphate phosphohydrolase activity as a 6.5 S multifunctional enzyme complex which contains two major polypeptides of 96,000 and 26,000 molecular weight. The characteristics of the various enzymatic reactions catalyzed by this complex are described. The GTP-PPi exchange reaction of vaccinia guanylyltransferase affords a simple, sensitive assay for capping enzyme function. The relevance of the GTP-PPi exchange reaction to the mechanism of transguanylylation is considered.     

Blocking of targeted microRNAs from next-generation sequencing libraries

Highly abundant microRNAs (miRNAs) in small RNA sequencing libraries make it difficult to obtain efficient measurements of more lowly expressed species. We present a new method that allows for the selective blocking of specific, abundant miRNAs during preparation of sequencing libraries. This technique is specific with little off-target effects and has no impact on the reproducibility of the measurement of non-targeted species. In human plasma samples, we demonstrate that blocking of highly abundant hsa-miR-16–5p leads to improved detection of lowly expressed miRNAs and more precise measurement of differential expression overall. Furthermore, we establish the ability to target a second abundant miRNA and to multiplex the blocking of two miRNAs simultaneously. For small RNA sequencing, this technique could fill a similar role as do ribosomal or globin removal technologies in messenger RNA sequencing.     

The fractionation of dinucleoside monophosphate and some trinucleoside diphosphate isonicotinoyl hydrazones by column chromatography

A column-chromatographic system using DEAE-cellulose and gradient elution with triethylammonium formate at pH4.0-3.5 is described. It is capable of separating the oligonucleotide isonicotinoyl hydrazones that are produced by nuclease digestion of RNA oxidized with periodate and coupled with isonicotinic acid hydrazide. Fifteen dinucleoside monophosphate isonicotinoyl hydrazones were characterized by their elution positions on the columns, so that all but two of them could readily be identified. Twelve trinucleoside diphosphate hydrazones were also characterized by their elution positions on the column. The application of this method of fractionation to terminal-sequence studies of RNA is discussed.     

A New Solid-Phase Chemical DNA Sequencing Method Which Uses Streptavidin-Coated Magnetic Beads

To simplify the chemical DNA sequencing protocol, we developeda new solid-phase method which uses streptavidin-coated magneticbeads. This method is based on the finding that the biotinylatedDNA-streptavidin complex was stable under the conditions forsome chemical sequencing reactions. The 5'-biotinylated DNAgenerated by the polymerase chain reaction was first capturedby streptavidin-coated magnetic beads and then subjected toa set of simplified chemical sequencing reactions on the beadsat room temperature. Followed by the piperidine cleavage reaction,the products were resolved by gel electrophoresis, transferredonto a nylon membrane and visualized by chemiluminescent detection.As a consequence, highquality sequencing ladders were obtained,due to complete removal of contaminating chemicals, withoutthe time-consuming precipitation/centrifugation steps used inthe conventional chemical sequencing protocol     

Fluorescence detection of amino acids in the postcleavage conversions for manual sequencing of a peptide

A modified Edman degradation method where fluorescent derivatives of amino acids were generated from the postcleavage products of a peptide is described. In the method, the target peptide was applied onto double glass fiber membranes in a small filter disk (4 mm i.d.) and then treated with small amounts of reagents for the manual sequencing of the peptide. The anilinothiazolinone (ATZ) of N-terminus amino acid residue after the isolation from the solid-phase membranes was reacted with a primary amine, 4-(1′-cyanoisoindolyl)aniline (CIA), to form a more stable and sensitive fluorescent derivative, phenylthiocarbamoyl-CIA. An average yield of 85% was obtained in neutral pH conditions for the CIA reaction. The ATZ-CIA-amino acids were separated by reversed-phase liquid chromatography and detected by fluorometry. The lower limits of the detection for amino acids after the Edman degradation were 0.16 to 0.52 pmol (signal/noise ratio = 3) on the column. The sensitivity was approximately 10 times higher than ultraviolet absorbance detection of phenylthiohydantoin products in the conventional Edman degradation. The suitability of the method was demonstrated by the sensitive manual sequencing of insulin chain B composed of 30 amino acids.     

Solid phase DNA sequencing using the biotin-avidin system.

A novel method for solid-phase DNA sequencing is described. A plasmid vector, pRIT27, has been designed to allow directional immobilization of double stranded plasmid to avidin agarose. The strategy involves enzymatic incorporation of 11-bio-dUTP into the plasmid and strand specific elution using alkali. The immobilized single stranded DNA is used as template for sequencing reactions and the resulting labelled oligonucleotides are eluted by alkali. The affinity gel containing the immobilized template is consecutively used for the four different dideoxy-nucleotide reactions. The solid-phase technique can be used for both primer specific or extension specific labelling. The possibility to use the system in automated DNA sequencing is discussed.     

Purification and analysis of the major components of chum salmon protamine contained in insulin formulations using high-performance liquid chromatography

A simple high-performance liquid chromatographic method has been developed for the rapid purification and analysis of protamine components contained in insulin formulations. Only a single step is needed to separate peptides whose compositions, sizes, and unusual isoelectric points (pI 13.8) are nearly identical. The method involves their isocratic separation on a reversed-phase column using a pH 2 phosphate buffer and a low acetonitrile content as an eluant. The purified chum salmon components were analyzed by amino acid analysis, solid-phase amino acid sequencing, carboxypeptidase B digests, insulin complexation analysis, and a mass spectrophotometric procedure which gives an accurate mass of the intact peptides. This HPLC purification technique may also be applicable to protamines and other highly basic peptides isolated from other sources.     

Solid-phase assay for determination of binding parameters of ligand-protein complexes with high dissociation rates

The binding parameters, the affinity constant (Ka) and binding capacity (Q), of a protein possessing ligand-protein complexes with a high dissociation rate (Sex Steroid Binding protein from Bufo arenarum) were determined using a solid-phase method. The technique is based upon the adsorption of the steroid-protein complex to DEAE-cellulose. This method was compared with a nonequilibrium method (charcoal adsorption of free ligand), and the latter resulted in underestimation of both binding parameters, Ka and Q. The solid-phase method reported here is appropriate to determine the binding parameters of proteins with high dissociation rates because the results are independent of the complex half-time. The method also possesses advantages compared to other equilibrium assays such as dialysis or steady-state electrophoresis. With minor modifications, it may be useful to characterize different proteins, particularly those possessing ligand-protein complexes with very high dissociation rates.     

利用Dynabeads固相单链分离法直接测定PCR产物序列

用PCR方法扩增了低密度脂蛋白(LDL)受体基因外显子11-内含子11-外显子12片段,长约0.8kb。利用Dynabeads固相分离单链法测定了PCR产物序列,其中有105个碱基系第一次报道,结果证明:Dynabeads固相分离单链法是一种简便、可靠、快速的PCR产物测序法。     

Simple and efficient synthesis of 5' pre-adenylated DNA using thermostable RNA ligase

We report a simple method of enzymatic synthesis of pre-adenylated DNA linkers/adapters for next-generation sequencing using thermostable RNA ligase from Methanobacterium thermoautotrophicum (MthRnl). Using RNA ligase for the reaction instead of the existing chemical or T4 DNA ligase-based methods allows quantitative conversion of 5'-phosphorylated single-stranded DNA (ssDNA) to the adenylated form. The MthRnl adenylation reaction is specific for ATP and either ssDNA or RNA. In the presence of Mg(+2), the reaction has a pH optimum of 6.0-6.5. Unlike reactions that use T4 DNA ligase, this protocol does not require synthesis of a template strand for adenylation. The high yield of the reaction simplifies isolation and purification of the adenylated product. Conducting the adenylation reaction at the elevated temperature (65°C) reduces structural constraints, while increased ATP concentrations allow quantitative adenylation of DNA with a 3'-unprotected end.     

Gradient thin-layer chromatography of oligonucleotides on DEAE-cellulose: an alternative to homochromatography.

Homochromatography fingerprints are widely used for sequence studies of labeled RNA: however, the more general use of this method is restricted in several aspects by the large excess of RNA introduced by the homomix. Gradient thin-layer chromatography on DEAE-cellulose plates using ammonium formate gradients in 9 m urea provides an efficient procedure for preparing fingerprints of labeled as well as unlabeled RNA, and allows the isolation of oligonucleotides free of salt, urea, and carrier RNA. This method produces fingerprints similar to those obtained by homochromatography under appropriate conditions. Consequently, gradient thin-layer chromatography is a convenient alternative to homochromatography without some of its limitations.