Applications of topologically segregated bilayer beads in 'one-bead one-compound' combinatorial libraries.

We recently reported the use of a biphasic approach to generate topologically segregated bilayer beads. In generating 'one-bead one-compound' (OBOC) combinatorial libraries, novel encoding methods have been applied to these beads such as the testing library compound and the coding tags residing on the outer layer and inner core of each bead, respectively. In this report, we further exploit these bilayer beads by preparing target bead-libraries with low concentration of random peptides on the outer layer, and full substitution of coding peptides in the bead interior. The low concentration of peptide on the bead surface enables us to greatly increase the stringency of screening so that higher affinity ligands can easily be identified. Full substitution of the inner core of the beads enables us to have enough coding peptides inside the bead for direct microsequencing with Edman chemistry. The biphasic approach of preparing bilayer beads can be carried out at any point during the library construction. Therefore, the nonsequencable or fixed structures of the peptides can be bypassed in the coding tags. As a result, peptide libraries that otherwise cannot be sequenced can now be sequenced, and peptide segments with fixed residues within the libraries can be bypassed so that the microsequencing time can be significantly shortened. Furthermore, peptides with a branch of random sequence in the middle of a fixed peptide chain can be encoded with just the random sequence in the bead interior. We have successfully applied these novel OBOC library concepts in the optimization of cell-surface ligands for a human T-cell leukemia, Jurkat, cell line.     

5'-Terminal cap structures of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells: comparison with other ribonucleic acid subpopulations

The 5'-terminal cap structures of 32P-labeled oligo(uridylic acid)-containing messenger ribonucleic acid [oligo(U+)mRNA] isolated from HeLa cell polyadenylated [poly(A+)] mRNA were analyzed and compared to those of the poly(A+) mRNA. A method employing P1 nuclease, alkaline phosphatase, and adsorption to activated charcoal showed that the types of cap core (m7 GpppXm) in oligo(U+) mRNA were essentially identical with those in poly(A+) mRNA. Analysis of RNase T2 digestion products of oligo(U+) mRNA demonstrated the presence of both cap 1 (m7GpppXmpYp) and cap 2 (m7GpppXmpYmpZp) in this subpopulation, confirming its cytoplasmic location. The base compositions of these two types of caps were different from each other and nonrandom but did not differ significantly between oligo(U+) and poly(A+) m RNA. The only observed difference between the mRNA populations was a higher ratio of cap 1 and cap 2 in the former. Possible implications of these findings for the relationship between oligo(U+) mRNA and poly(A+) mRNA are discussed.     

Optimizing the immobilization of single-stranded DNA onto glass beads

The attachment of single-stranded DNA to a solid support has many biotechnology and molecular biology applications. This paper compares different immobilization chemistries to covalently link single-stranded DNA (20 base pairs), oligo(1), onto glass beads via a 5'-amino terminal end. Immobilization methods included a one-step 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and a two-step EDC reaction to succinylated and PEG-modified glass beads. The third method used 1,4-phenylene diisothiocyanate to immobilize oligo(1) to aminopropyl glass beads. The influence of coupling buffer, oligo(1) concentration, and EDC concentration was also investigated. The one-step EDC-mediated procedure with succinylated or PEG-modified beads in 0.1 M MES buffer, pH 4.5, resulted in the highest immobilization efficiency, 82-89%. EDC concentrations greater than 50 mM and oligo(1) concentrations of 3 microg/g bead were required for effective immobilization. A complementary oligonucleotide, oligo(2), was able to hybridize to the immobilized oligo(1) with a 58% efficiency. This oligonucleotide was subsequently released at 70 degrees C. The relationship between the surface density of oligo(1) and the hybridization efficiency of the complementary oligonucleotide is described.     

Identification of differentially expressed genes by mutually subtracted RNA fingerprinting

A mutually subtracted RNA fingerprinting (SuRF) method has been developed that allows efficient identification of differentially expressed sequence tags between two samples. Mutual subtractions of two RNA samples are achieved by first synthesizing cDNAs using oligo(dT) coupled with magnetic beads which are then reciprocally hybridized to starting RNA samples to remove common mRNAs between them. The second step involves differential fingerprinting of the subtracted RNA samples by polymerase chain reaction with specially designed degenerate primers. SuRF was applied to identify alteration in gene expression pertinent to osteogenic sarcoma which was achieved by employing the method between FOB (an immortalized fetal osteoblast) and MG63 (an osteosarcoma) cell lines. An estimated 10% of the total expressed genes in these two cell types were screened by the method. This analysis identified 96 differentially expressed sequences, none of which was identified repeatedly. A subset of these sequences was subsequently confirmed to have differential expression between the two cell types. Removal of common mRNAs prior to differential display should diminish redundant identification of abundant genes and increase the chance of identifying rare differentially expressed genes.     

DNA probes on beads arrayed in a capillary, 'Bead-array', exhibited high hybridization performance

A DNA analysis platform called 'Bead-array' is presented and its features when used in hybridization detection are shown. In 'Bead-array', beads of 100- micro m diameter are lined in a determined order in a capillary. Each bead is conjugated with DNA probes, and can be identified by its order in the capillary. This probe array is easily produced by just arraying beads conjugated with probes into the capillary in a fixed order. The hybridization is also easily completed by introducing samples (1-300 micro l) into the capillary with reciprocal flow. For hybridization detection, as little as 1 amol of fluorescent-labeled oligo DNA was detected. The hybridization reaction was completed in 1 min irrespective of the amount of target DNA. When the number of target molecules was smaller than that of probe molecules on the bead, 10 fmol, almost all targets were captured on the bead. 'Bead-array' enables reliable and reproducible measurement of the target quantity. This rapid and sensitive platform seems very promising for various genetic testing tasks.     

Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library

Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA.     

Pooled library tissue tags for EST-based gene discovery

MOTIVATION: In gene discovery projects based on EST sequencing, effective post-sequencing identification methods are important in determining tissue sources of ESTs within pooled cDNA libraries. In the past, such identification efforts have been characterized by higher than necessary failure rates due to the presence of errors within the subsequence containing the oligo tag intended to define the tissue source for each EST. RESULTS: A large-scale EST-based gene discovery program at The University of Iowa has led to the creation of a unique software method named UITagCreator usable in the creation of large sets of synthetic tissue identification tags. The identification tags provide error detection and correction capability and, in conjunction with automated annotation software, result in a substantial improvement in the accurate identification of the tissue source in the presence of sequencing and base-calling errors. These identification rates are favorable, relative to past paradigms. AVAILABILITY: The UITagCreator source code and installation instructions, along with detection software usable in concert with created tag sets, is freely available at http://genome.uiowa.edu/pubsoft/software.html CONTACT: tomc@eng.uiowa.edu     

A simple and versatile method for the preparation of vector-primers by adapter-end-primer ligation

A group of efficient cDNA cloning strategies employs vector-primers where cDNA synthesis starts from the oligo(dT)-primer tail, which is conventionally attached to cloning vectors by use of terminal deoxynucleotidyl transferase. An alternative, efficient and more versatile method of vector-primer preparation is to directly ligate, by use of T4 DNA ligase, a double-digested vector, e.g., pTZ18R/Pst I/Bam HI, to a synthetic (Bam HI)-adapter-end-primer, 5'-pGATCC-Tn or 5'-pGATCC-site-specific sequence. The use of a utility-vector containing a sizable spacer between the two selected restriction sites enables unambiguous separation on agarose gels of the double-digested vector precursors from single-digested ones, further simplifying the vector preparation. The adapter-end-primer ligation method can be applied to any suitable vectors with multiple cloning sites for the preparation of not only oligo(dT)-tailed, but also site-specific sequence-tailed vectors. Thus, the method enables the cDNA cloning of total poly (A+)-mRNAs, as well as specific RNA or mRNA species with or without poly(A)-tail.     

Evaluation of high-performance anion-exchange chromatography with pulsed electrochemical and fluorometric detection for extensive application to the analysisof homologous series of oligo- and polysialic acids in bioactive molecules.

Our previous studies have shown extensively diverse structures in oligo/polymers of sialic acid (oligo/polySia) that are expressed often in developmentally regulated manner on animal glycoconjugates. The aim of this study was to establish highlysensitive and specific methods that can be used to identify diverse types of oligo/polySia and thus can be applied to studies of biological phenomena associated with the differential expression of oligo/polySia chains with different degree of polymerization (DP). As model compounds, we analyzed five different homologous series of oligo/polySia, (-->8Neu5Acalpha2-->)(n), (-->9Neu 5Acalpha2-->)(n), (-->8Neu5Gcalpha2-->)(n), (-->5-O(glycolyl)-Neu5Gcalpha2-->)(n), and Neu5Gc9SO(4)alpha2-->(-->5-O(glycolyl)-Neu5Gcalpha2--> )(n), ()expressed in various biopolymers. The latter two structures have recently been identified in sea urchin egg receptor for sperm. First we examined application of high-performance anion-exchange chromatography (HPAEC) on a CarboPac PA-100 column with pulsed electrochemical detection (PED) to new types of oligo/polySiacompounds and confirmed that resolution of high polymers (DP >70) of sialic acids was remarkable as reported previously. However, there are limitations in sensitivity and selectivity in PED that become significant when material is available only in a minute amount or material contained a large proportion of protein. These limitations can be circumvented by fluorometric detection of oligo/polySia tagged with 1,2-diamino-4, 5-methyl-enedioxybenzene (DMB) at the reducing terminal residues after separation on a MonoQ HR5/5 column. The latter method can be applied to any type of oligo/polySia we examined if we choose the derivatization conditions and is more sensitive and specific than the method with PED for analysis of oligo/polySia with DP up to 25.     

Construction of a large signature-tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti

Sinorhizobium meliloti genome sequence determination has provided the basis for different approaches of functional genomics for this symbiotic nitrogen-fixing alpha-proteobacterium. One of these approaches is gene disruption with subsequent analysis of mutant phenotypes. This method is efficient for single genes; however, it is laborious and time-consuming if it is used on a large scale. Here, we used a signature-tagged transposon mutagenesis method that allowed analysis of the survival and competitiveness of many mutants in a single experiment. A novel set of signature tags characterized by similar melting temperatures and G+C contents of the tag sequences was developed. The efficiencies of amplification of all tags were expected to be similar. Thus, no preselection of the tags was necessary to create a library of 412 signature-tagged transposons. To achieve high specificity of tag detection, each transposon was bar coded by two signature tags. In order to generate defined, nonredundant sets of signature-tagged S. meliloti mutants for subsequent experiments, 12,000 mutants were constructed, and insertion sites for more than 5,000 mutants were determined. One set consisting of 378 mutants was used in a validation experiment to identify mutants showing altered growth patterns.     

A One-Bead One-Peptide Combinatorial Library Method for B-Cell Epitope Mapping

The one-bead one-peptide combinatorial library method represents a powerful approach to the discovery of binding peptides for various macromolecular targets. It involves the synthesis of millions of peptides on beads such that each bead displays only one peptide entity. The peptide–beads that interact with a specific macromolecular target are then isolated for structure determination. We have applied this method to discovering peptide ligands for several murine monoclonal antibodies: (i) anti-β-endorphin (continuous epitope), (ii) anti-vmos peptide, (iii) anti-human insulin (discontinuous epitope), and (iv) surface immunoglobulins (μκ) of two murine B-cell lymphoma cell lines (antigen unknown).     

DNA probes on beads arrayed in a capillary, ‘Bead-array’, exhibited high hybridization performance

A DNA analysis platform called ‘Bead-array’ is presented and its features when used in hybridization detection are shown. In ‘Bead-array’, beads of 100-µm diameter are lined in a determined order in a capillary. Each bead is conjugated with DNA probes, and can be identified by its order in the capillary. This probe array is easily produced by just arraying beads conjugated with probes into the capillary in a fixed order. The hybridization is also easily completed by introducing samples (1–300 µl) into the capillary with reciprocal flow. For hybridization detection, as little as 1 amol of fluorescent-labeled oligo DNA was detected. The hybridization reaction was completed in 1 min irrespective of the amount of target DNA. When the number of target molecules was smaller than that of probe molecules on the bead, 10 fmol, almost all targets were captured on the bead. ‘Bead-array’ enables reliable and reproducible measurement of the target quantity. This rapid and sensitive platform seems very promising for various genetic testing tasks.     

DNA-directed coupling of organic modules by multiple parallel reductive aminations and subsequent cleavage of selected DNA sequences

A new method for DNA-directed assembly of organic modules by multiple parallel reductive aminations is presented. Linear oligonucleotide-functionalized modules (LOMs) consist of a rigid oligo(phenylene ethynylene) backbone with two salicylaldehyde termini, and each terminus is conjugated with an oligonucleotide sequence. The stability of the tetrahydrosalen-linked modules toward elevated temperature, low pH, nucleophiles, and metal chelators is studied and compared to the analogous metal-salen-linked modules. A linear oligonucleotide-functionalized disulfide-linked module (LOSM) containing cleavable linkers between the organic module and the two DNA sequences is coupled by DNA-directed reductive aminations to non-modified LOM modules. This enables selective cleavage of the DNA strands of a central module in a structure consisting of three modules, and the reactions are analyzed by electrophoresis and 32P-labeling of one of the DNA sequences of the central LOSM.     

Construction of a Large Signature-Tagged Mini-Tn5 Transposon Library and Its Application to Mutagenesis of Sinorhizobium meliloti

Sinorhizobium meliloti genome sequence determination has provided the basis for different approaches of functional genomics for this symbiotic nitrogen-fixing alpha-proteobacterium. One of these approaches is gene disruption with subsequent analysis of mutant phenotypes. This method is efficient for single genes; however, it is laborious and time-consuming if it is used on a large scale. Here, we used a signature-tagged transposon mutagenesis method that allowed analysis of the survival and competitiveness of many mutants in a single experiment. A novel set of signature tags characterized by similar melting temperatures and G+C contents of the tag sequences was developed. The efficiencies of amplification of all tags were expected to be similar. Thus, no preselection of the tags was necessary to create a library of 412 signature-tagged transposons. To achieve high specificity of tag detection, each transposon was bar coded by two signature tags. In order to generate defined, nonredundant sets of signature-tagged S. meliloti mutants for subsequent experiments, 12,000 mutants were constructed, and insertion sites for more than 5,000 mutants were determined. One set consisting of 378 mutants was used in a validation experiment to identify mutants showing altered growth patterns.     

Seahorse fingerprints: a new individual identification technique

Individual identification is particularly important for ethological studies and sampling design. Previous studies have developed various types of marking by tags and chemical marks, but these methods involve significant manipulation of the individuals. Other studies have reported natural marks as an efficient method for individual recognition. Our study aims to elucidate a new method for natural mark identification of seahorses, which we tested with the endangered Brazilian seahorse Hippocampus reidi. We avoid pseudoreplication by recognizing individuals. Seahorses have a hard bony structure on the top of their heads called the coronet, which has a different shape for each individual, corresponding to a fingerprint. We tagged seahorses in the field with collar tags and photographed their coronets. After two days, we identified seahorses by their photographs and verified their identification with the collar tags. We correctly identified all individuals by fingerprint identification. Although this method was only tested with adults, we suggest that it applies to seahorses in general, as all species possess the coronet structure.     

Evaluation of different methods for the extraction of DNA from fungal conidia by quantitative competitive PCR analysis.

Five different DNA extraction methods were evaluated for their effectiveness in recovering PCR templates from the conidia of a series of fungal species often encountered in indoor air. The test organisms were Aspergillus versicolor, Penicillium chrysogenum, Stachybotrys chartarum, Cladosporium herbarum and Alternaria alternata. The extraction methods differed in their use of different cell lysis procedures. These included grinding in liquid nitrogen, grinding at ambient temperature, sonication, glass bead milling and freeze-thawing. DNA purification and recovery from the lysates were performed using a commercially available system based on the selective binding of nucleic acids to glass milk. A simple quantitative competitive polymerase chain reaction (QC-PCR) assay was developed for use in determining copy numbers of the internal transcribed spacer (ITS) regions of the ribosomal RNA operon (rDNA) in the total DNA extracts. These quantitative analyses demonstrated that the method using glass bead milling was most effective in recovering PCR templates from each of the different types of conidia both in terms of absolute copy numbers recovered and also in terms of lowest extract to extract variability. Calculations of average template copy yield per conidium in this study indicate that the bead milling method is sufficient to support the detection of less than ten conidia of each of the different organisms in a PCR assay.     

A systematic, ligation-based approach to study RNA modifications

Over 100 different chemical types of modifications have been identified in thousands of sites in tRNAs, rRNAs, mRNAs, small nuclear RNAs, and other RNAs. Some modifications are highly conserved, while others are more specialized. They include methylation of bases and the ribose backbone, rotation, and reduction of uridine, base deamination, elaborate addition of ring structures, carbohydrate moieties, and more. We have developed a systematic approach to detect and quantify the extent of known RNA modifications. The method is based on the enzymatic ligation of oligonucleotides using the modified or unmodified RNA as the template. The efficiency of ligation is very sensitive to the presence and the type of modifications. First, two oligo pairs for each type of modification are identified. One pair greatly prefers ligation using the unmodified RNA template over the modified RNA template or vice versa. The other pair has equal reactivity with unmodified and modified RNA. Second, separate ligations with each of the two oligo pairs and the total RNA mixture are performed to detect the presence or absence of modifications. Multiple modification sites can be examined in the same ligation reaction. The feasibility of this method is demonstrated for three 2'O-methyl modification sites in yeast rRNA.