High-Density Microarray of Small-Subunit Ribosomal DNA Probes

Ribosomal DNA sequence analysis, originally conceived as a way to provide a universal phylogeny for life forms, has proven useful in many areas of biological research. Some of the most promising applications of this approach are presently limited by the rate at which sequences can be analyzed. As a step toward overcoming this limitation, we have investigated the use of photolithography chip technology to perform sequence analyses on amplified small-subunit rRNA genes. The GeneChip (Affymetrix Corporation) contained 31,179 20-mer oligonucleotides that were complementary to a subalignment of sequences in the Ribosomal Database Project (RDP) (B. L. Maidak et al., Nucleic Acids Res. 29:173-174, 2001). The chip and standard Affymetrix software were able to correctly match small-subunit ribosomal DNA amplicons with the corresponding sequences in the RDP database for 15 of 17 bacterial species grown in pure culture. When bacteria collected from an air sample were tested, the method compared favorably with cloning and sequencing amplicons in determining the presence of phylogenetic groups. However, the method could not resolve the individual sequences comprising a complex mixed sample. Given these results and the potential for future enhancement of this technology, it may become widely useful.     

Fluorescent Labeling of Oligonucleotide Probes for Double Indicator Microarray Hybridization Analysis

Russian Journal of Bioorganic Chemistry - Herein, we present a synthesis of a molecular construct based on the polysulfonated indocarbocyanine dye and the lysine. The chemical structure of the...     

Improved In Situ Hybridization Efficiency with Locked-Nucleic-Acid-Incorporated DNA Probes

Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.     

Nonradioactive in Situ Hybridization with Digoxigenin Labeled DNA Probes

Nonradioactive in situ hybridization techniques are becoming increasingly important tools for rapid analysis of the topological organization of DNA and RNA sequences within cells. Prerequisite for further advances with these techniques are multiple labeling and detection systems for different probes. Here we summarize our results with a recently developed labeling and detection system. The DNA probe for in situ hybridization is modified with digoxigenin-labeled deoxyuridine-triphosphate. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Labeled DNA probes were hybridized in situ to chromosome preparations. The hybridization signal was detected using digoxigenin-specific antibodies covalently coupled to enzyme markers (alkaline phosphatase or peroxidase) or to fluorescent dyes. Color reactions catalyzed by the enzymes resulted in precipitates located on the chromosomes at the site of probe hybridization. This was verified by hybridizing DNA probes of known chromosomal origin. The signals were analyzed by bright field, reflection contrast and fluorescence microscopy. The results indicate that the new technique gives strong signals and can also be used in combination with other systems (e.g., biotin) to detect differently labeled DNA probes on the same metaphase plate.     

DNA Probes: Applications of the Principles of Nucleic Acid Hybridization

Abstract

Nucleic acid hybridization with a labeled probe is the only practical way to detect a complementary target sequence in a complex nucleic acid mixture. The first section of this article covers quantitative aspects of nucleic acid hybridization thermodynamics and kinetics. The probes considered are oligonucleotides or polynucleotides, DNA or RNA, single- or double-stranded, and natural or modified, either in the nucleotide bases or in the backbone. The hybridization products are duplexes or triplexes formed with targets in solution or on solid supports. Additional topics include hybridization acceleration and reactions involving branch migration. The second section deals with synthesis or biosynthesis and detection of labeled probes, with a discussion of their sensitivity and specificity limits. Direct labeling is illustrated with radioactive probes. The discussion of indirect labels begins with biotinylated probes as prototypes. Reporter groups considered include radioactive, fluorescent, and chemiluminescent nucleotides, as well as enzymes with colorimetric, fluorescent, and luminescent substrates.     

Detect the Hybridization of Single-Stranded DNA by Parallel Scan Spectral Surface Plasmon Resonance Imaging

We investigate the ability of a parallel scan spectral surface plasmon resonance imaging system in analyzing hybridization of single-stranded DNA (ssDNA) on microarray. The ssDNA probes are modified by a thiol group and thereby can be immobilized onto the gold film. Hybridization experiments are carried out by using both complementary and noncomplementary sequence to confirm the specificity of interaction. We also demonstrate that the data analysis is very important in reducing the noise and improving the resolution by comparing polynomial fitting method with the combination of polynomial fitting and centroid method. Finally, the results demonstrate that the parallel scan spectral surface plasmon resonance imaging system can be used for high-throughput analysis of the hybridization of the ssDNA.     

Detection and Classification of Trypanosoma cruzi by DNA Hybridization with Nonradioactive Probes

ABSTRACT. Total or kinetoplast DNA (kDNA) from 72 isolates and clones of Trypanosoma cruzi as well as from nine related trypanosomatids were analyzed by dot hybridization using nonradioactive kDNA or cloned minicircle fragments as probes. Biotinylated-kDNA probes generated by nick-translation proved reliable for distinguishing Zymodeme 1 and Zymodeme 2bol of T. cruzi parasites. In contrast, digoxigenin-labeled kDNA obtained by random-priming did not distinguish among T. cruzi isolates but did distinguish among New World leishmanias. Cloned minicircle fragments labeled with digoxigenin gave the same results as digoxigenin-labeled kDNA, except for a 10-fold decrease in sensitivity. Digoxigenin-labeled DNA probes proved useful in unambiguously detecting T. cruzi from different geographic regions of America. However, T. rangeli and T. cruzi marinkellei were not distinguished by these probes.     

Construction of Europium (Eu3+)-Labelled Oligo DNA Hybridization Probes

Abstract

Three different oligo DNA probes were synthesized and their value as hybridization probes was tested.     

Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens

In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes loci dropout. This should enable the application of hyRAD to analyses at broader evolutionary scales.     

苏云金杆菌DNA芯片Oligo探针设计

迅速增长的分子生物学数据为总结新的生物学信息,进行生物研究尤其是分子生物学研究,提供了丰富的资料,利用苏云金杆菌的基因信息和一些生物学软件,设计特异性高、长度一致、熔解温度相近的Oligo探针,为后期打印成DNA芯片,进行苏云金杆菌鉴定打下基础。     

炭疽芽胞杆菌基因芯片探针文库的构建

为制备炭疽芽胞杆菌的基因芯片探针文库,以炭疽芽胞杆菌毒素质粒pX01和荚膜质粒pX02为原材料,用Sau3A I酶切pX01和pX02质粒DNA,Taq DNA聚合酶72℃补平加A,经AT克隆,PCR初步鉴定筛选出炭疽质粒片段的阳性克隆．DNA自动分析仪对克隆片段进行序列测定;用生物信息学方法对其片段进行同源性分析;并将克隆的探针打印于玻片上,制备成炭疽芽胞杆茵基因芯片,与炭疽杆茵质粒DNA样品进行初步芯片杂交的实验,杂交实验的阳性率达到了90％以上,证明大部分克隆探针属于炭疽芽胞杆菌．炭疽芽胞杆菌基因芯片探针文库的构建为基因芯片探针的制备摸索出一条简便、高效、可行的方法．     

用巢式PCR方法制备基因芯片的探针

制备出高纯度的探针,用于诊断基因芯片的打印。采用巢式PCR技术,M13作为外侧引物并自行设计内侧引物,扩增克隆在T载体上的基因片段。可制备出成分单一,上下游仅含19bp和20bp的短载体序列的探针,能使打印出的芯片得到较好的杂交效果。该法充分利用巢式PCR的优点,对制备探针的方法进行改进,且简便快速,能得到更高质量的探针,满足打印芯片的要求。     

Reverse in Situ Hybridization of DNA Probes of Abnormal Chromosomes in Diagnostics of Chromosomal Pathologies

The results of analysis of congenital chromosomal pathologies and chromosomal rearrangements upon the occurrence of haematological diseases, which was involved constructing DNA libraries of abnormal chromosomes and subsequent reverse CISS hybridization have been considered. High effectiveness of this approach for analysis of chromosomal translocations, deletions of chromosomal regions, minor additional chromosomes, and large marker chromosomes with complex organization was shown. The possibility of implementation of this approach and its large-scale application in medical and genetic studies of congenital developmental pathologies and chromosomal diagnostics of haematological diseases has been discussed.     

Application of Equilibrium Models of Solution Hybridization to Microarray Design and Analysis

Background

The probe percent bound value, calculated using multi-state equilibrium models of solution hybridization, is shown to be useful in understanding the hybridization behavior of microarray probes having 50 nucleotides, with and without mismatches. These longer oligonucleotides are in widespread use on microarrays, but there are few controlled studies of their interactions with mismatched targets compared to 25-mer based platforms.

Principal Findings

50-mer oligonucleotides with centrally placed single, double and triple mismatches were spotted on an array. Over a range of target concentrations it was possible to discriminate binding to perfect matches and mismatches, and the type of mismatch could be predicted accurately in the concentration midrange (100 pM to 200 pM) using solution hybridization modeling methods. These results have implications for microarray design, optimization and analysis methods.

Conclusions

Our results highlight the importance of incorporating biophysical factors in both the design and the analysis of microarrays. Use of the probe “percent bound” value predicted by equilibrium models of hybridization is confirmed to be important for predicting and interpreting the behavior of long oligonucleotide arrays, as has been shown for short oligonucleotide arrays.     

Formation of Single-Stranded DNA during DNA Transformation of Neisseria gonorrhoeae

Neisseria gonorrhoeae is naturally competent for DNA transformation. In contrast to other natural prokaryotic DNA transformation systems, single-stranded donor DNA (ssDNA) has not previously been detected during transformation of N. gonorrhoeae. We have reassessed the physical nature of gonococcal transforming DNA by using a sensitive nondenaturing native blotting technique that detects ssDNA. Consistent with previous analyses, we found that the majority of donor DNA remained in the double-stranded form, and only plasmid DNAs that carried the genus-specific DNA uptake sequence were sequestered in a DNase I-resistant state. However, when the DNA was examined under native conditions, S1 nuclease-sensitive ssDNA was identified in all strains tested except for those bacteria that carried the dud-1 mutation. Surprisingly, ssDNA was also found during transformation of N. gonorrhoeae comA mutants, which suggested that ssDNA was initially formed within the periplasm.     

Comparison of Comparative Genomic Hybridization Technologies Across Microarray Platforms

In the 2007 Association of Biomolecular Resource Facilities Microarray Research Group project, we analyzed HL-60 DNA with five platforms: Agilent, Affymetrix 500K, Affymetrix U133 Plus 2.0, Illumina, and RPCI 19K BAC arrays. Copy number variation was analyzed using circular binary segmentation (CBS) analysis of log ratio scores from four independently assessed hybridizations of each platform. Data obtained from these platforms were assessed for reproducibility and the ability to detect formerly reported copy number variations in HL-60. In HL-60, all of the tested platforms detected genomic DNA amplification of the 8q24 locus, trisomy 18, and monosomy X; and deletions at loci 5q11.2~q31, 9p21.3~p22, 10p12~p15, 14q22~q31, and 17p12~p13.3. In the HL-60 genome, at least two of the five platforms detected five novel losses and five novel gains. This report provides guidance in the selection of platforms based on this wide-ranging evaluation of available CGH platforms.