A reproducible microanalytical method for the detection of specific RNA sequences by dot-blot hybridization

A rapid, microanalytical procedure for the reproducible isolation of RNA from small cultured cell samples and application to dot-blot hybridization is described. The procedure employs guanidine hydrochloride solubilization of whole cells, disruption by syringing, and selective precipitation of RNA with ethanol. The method can be performed in a single tissue culture tube and obviates the need for removal of nuclei or for organic solvent extractions. Recovery of RNA from small cell samples (10(6) cells) is 51%, while 97% of the DNA and 99% of the protein are eliminated by the procedure. Detection of specific RNA by dot-blot hybridization using a labeled probe demonstrates high reproducibility of recovered RNA and lack of "masking" with up to a 10-fold excess of starting cell material. Applicability of the procedure to detection of virus-specific RNA in cells persistently infected with mouse hepatitis virus is described.     

A new method that simultaneously aligns and reconstructs ancestral sequences for any number of homologous sequences, when the phylogeny is given

Among the fundamental problems in molecular evolution and in the analysis of homologous sequences are alignment, phylogeny reconstruction, and the reconstruction of ancestral sequences. This paper presents a fast, combined solution to these problems. The new algorithm gives an approximation to the minimal history in terms of a distance function on sequences. The distance function on sequences is a minimal weighted path length constructed from substitutions and insertions-deletions of segments of any length. Substitutions are weighted with an arbitrary metric on the set of nucleotides or amino acids, and indels are weighted with a gap penalty function of the form gk = a + (bxk), where k is the length of the indel and a and b are two positive numbers. A novel feature is the introduction of the concept of sequence graphs and a generalization of the traditional dynamic sequence comparison algorithm to the comparison of sequence graphs. Sequence graphs ease several computational problems. They are used to represent large sets of sequences that can then be compared simultaneously. Furthermore, they allow the handling of multiple, equally good, alignments, where previous methods were forced to make arbitrary choices. A program written in C implemented this method; it was tested first on 22 5S RNA sequences.      

An approach of orthology detection from homologous sequences under minimum evolution

In the field of phylogenetics and comparative genomics, it is important to establish orthologous relationships when comparing homologous sequences. Due to the slight sequence dissimilarity between orthologs and paralogs, it is prone to regarding paralogs as orthologs. For this reason, several methods based on evolutionary distance, phylogeny and BLAST have tried to detect orthologs with more precision. Depending on their algorithmic implementations, each of these methods sometimes has increased false negative or false positive rates. Here, we developed a novel algorithm for orthology detection that uses a distance method based on the phylogenetic criterion of minimum evolution. Our algorithm assumes that sets of sequences exhibiting orthologous relationships are evolutionarily less costly than sets that include one or more paralogous relationships. Calculation of evolutionary cost requires the reconstruction of a neighbor-joining (NJ) tree, but calculations are unaffected by the topology of any given NJ tree. Unlike tree reconciliation, our algorithm appears free from the problem of incorrect topologies of species and gene trees. The reliability of the algorithm was tested in a comparative analysis with two other orthology detection methods using 95 manually curated KOG datasets and 21 experimentally verified EXProt datasets. Sensitivity and specificity estimates indicate that the concept of minimum evolution could be valuable for the detection of orthologs.     

A rapid screen for the detection of specific DNA sequences in plants

We have developed a simple and quick method ("wick blot") for detecting the presence of specific DNA sequences in plants, using radiolabeled DNA probes. The method requires only small amounts of tissue, about 15-25 mg. More than a hundred samples per day can be easily extracted and blotted. It works well on various species and tissues, including leaves, embryos, and callus. The method is ideally suited for screening large numbers of putative transformants, especially populations that have not been screened by prior selection.     

Gene sequences and specific detection for Panton-Valentine leukocidin

A new category of methicillin-resistant Staphylococcus aureus (MRSA), called community-acquired MRSA (CA-MRSA), has emerged worldwide. In contrast to previous MRSA, most CA-MRSA carries the Panton-Valentine leukocidin (PVL) genes (lukPVSF) as a virulence genetic trait. Sequence analysis of the lukPVSF gene of a Japanese isolate demonstrated that the gene has more similarity to methicillin-susceptible S. aureus from France than MRSA from the United States. Based on the sequences, we developed a real-time PCR assay for the three key genes of CA-MRSA; that is, lukPVSF, mecA (for methicillin resistance), and spa (for S. aureus). Dual or triple assay for lukPVSF, mecA, and spa in one test tube became possible. The detection limit of the assay with probe and SYBR Green methods was between 2.7 and 2.7 x 10(1) CFU/ml. The assay detected PVL-positive MRSA in clinical (blood) isolates.     

A new method for the detection of alkane-monooxygenase homologous genes (alkB) in soils based on PCR-hybridization

An improved method was developed that allowed the specific detection of the gene alkB (coding for the rubredoxin dependent alkane monooxygenase) from bacteria without any obvious strain specific discrimination using a combination of PCR and hybridization. This approach enabled a fast culture-independent monitoring of environmental samples for the occurrence of alkB, and an estimation of the gene copy number and the genetic diversity. Both parameters provide useful informations for an assessment of the intrinsic biodegradation potential that is present at a site. The method was applied to soil samples from different uncontaminated sites. alkB was highly abundant and redundant in all soils tested. Potential biodegradation of n-alkanes was also demonstrated for these soils with substrate utilization assays. Cell numbers of hydrocarbon degraders estimated as MPN varied from 10(3) to 10(6)g(-1) soil (dry weight) for the different soils. Gene copy numbers estimated with MPN-PCR ranged within 1-40*10(4)ng(-1) soil DNA. Analysis of the diversity of the alkB sequences obtained from a grassland and an agricultural soil indicated that the alkane degrading microbial populations occurring at these sites were rather diverse. Compared on protein level, three major clusters were distinguishable for both soils that showed highest similarities to AlkB from the Gram-positives Nocardioides and Mycobacterium, and the Gram-negative Alcanivorax. The majority of the cloned AlkB sequences were homologous to proteins from the Gram-positive bacteria. However, significant differences from published sequences were observed; homologies varied from 50% to 90% (identity of amino acids).     

A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome-labelled RNA

A new method has been developed for the detection of in situ hybridization by fluorescence microscopy. It is based on the covalent binding of commercially available fluorochromes to the 3′-terminus of RNA.     

A general method for inserting specific DNA sequences into cloning vehicles

A general method has been developed to introduce any double-stranded DNA molecule into cloning vehicles at different restriction endonuclease sites. In this method a chemically synthesized decadeoxyribonucleotide duplex, containing a specific restriction endonuclease sequence, is joinlex DNA is cut by the same restriction endonuclease to generate the cohesive ends. It is then inserted into the restriction endonuclease cleavage site of the cloning vehicle. To demonstrate the feasibility of this new method, we have inserted separately the synthetic lac operator DNA at the Bam I and HindIII cleavage sites of the plasmid pMB9 DNA.     

A highly specific microarray method for point mutation detection

Improvements of microarray techniques for genotyping purposes have focused on increasing the reliability of this method. Here we report the development of a genotyping method where a microarray was spotted with stemloop probes, especially designed to optimize the hybridization specificity of complementary DNA sequences. This accurate method was used to screen for four common disease-causing mutations involved in a neurological disorder called Charcot-Marie-Tooth disease (CMT). Healthy individuals' and patients' DNA were amplified and labeled by PCR and hybridized on microarray. The spot signal intensities were 81 to 408 times greater for perfect compared with mismatched target sequences, differing by only one nucleotide (discrimination ratio) for healthy individual "homozygous" DNA. On the other hand, "heterozygous" mutant DNA samples gave rise to signal intensity ratios close to 1, as expected. The genotypes obtained by this method were perfectly consistent with those determined by direct PCR sequencing. Cross-hybridization rates were very low, resulting in further multiplexing improvements. In this study, we also demonstrated the feasibility of real-time hybridization detection of labeled synthetic oligonucleotides with concentrations as low as 2.5 nM.     

A new sensitive non-isotopic method using sulfonated probes to detect picogram quantities of specific DNA sequences on blot hybridization

The use of non-radioactive systems to detect target DNA or RNA displays many advantages such as safe manipulation, potential use in non-specialized scientific area and prolonged lifetime of the probes (one year or more). We here describe a method we have improved and optimized using sulfonated DNA probes for hybridization on dot and Southern blots. Sulfonation is an easy chemical modification procedure which does not require enzymatic coctail as does nick-translation. Sensitivity of this method has been particularly improved by using a new blocking solution, containing heparin, which allows easy and fast detection of picogram quantities of DNA. This method allows the use of nitrocellulose as well as nylon membranes with very low background. Equal resolution is obtained in comparative experiments involving both sulfonated and 32P-radiolabelled probes. Single copy gene sequences are readily detected in nuclear DNA. These results allow the use of this procedure for restriction fragment length polymorphism (RFLP) studies.     

Profile-profile比对方法用于发现远距离同源模板

基于模板的模建方法是蛋白质结构预测领域中最为准确有效的方法,该类方法的成功与否对模板质量的要求较高。为待预测序列找寻合适的模板,本文提出了一种profile-profile比对的方法将查询序列同模板库中的已知结构蛋白进行比对,然后根据比对结果的Z-score得分高低顺序挑选出合适的模板。结果表明:本文的profile-profile比对方法在测试集上的性能明显优于PSI-BLAST,相比PSI-BLAST在测试集上的准确度提高了约14.3%,配对t检验的结果表明准确度的提高具有统计显著性。从而得出如下结论:本文的profile-profile比对方法可以用于为序列相似性较低的待预测序列搜索远距离同源模板,并用于指导后续的三级结构预测。     

A colorimetric method for detection of specific ligand binding.

The binding of IL-2 and IL-3 to the factor-dependent cell lines CTB6 and 32D, respectively, was determined using biotinylated ligand detected by the addition of a streptavidin/alkaline phosphatase conjugate and amplified with a phosphatase amplification system. Binding of both ligands was detectable after incubation with as little as 20 fmol of ligand and could be inhibited with a 10-fold molar excess of nonbiotinylated ligand. No binding was observed when biotinylated ligand was incubated with a receptor negative cell line (PC-12) and IL-2 was unable to compete with biotinylated IL-3 binding to 32D cells, further demonstrating specificity. These studies indicate that biotinylated ligands can be used as a nonradioactive method to detect specific, high-affinity cell surface receptors.     

A quantitative method for analyzing specific DNA sequences directly from whole cells

A quick, accurate assay for specific DNA sequences is described in which whole cells are treated with 0.4 M sodium hydroxide at 80 degrees C. DNA is relatively resistant to alkaline hydrolysis, whereas proteins and RNA are degraded rapidly. The DNA in NaOH is then transferred through a slot directly onto a nylon membrane and hybridized with a probe. Since the procedure is so simple, many samples can be analyzed in a short time. A single-copy gene can be detected in as few as 1000 cells and, since the DNA from 10(5) cells can be loaded through a single slot, the sensitivity is sufficient to detect one specific DNA sequence per 100 cells. Accurate quantitative analysis can be achieved by normalizing the amount of DNA available for hybridization in each slot, using a probe derived from total DNA.     

A method of detecting conserved and variable segments in the amino acid sequences of homologous proteins

A set of aligned homologous protein sequences is divided into two groups consisting of m and n sequences. Each group contains sequences from the most related organisms. Value of the position dissimilarity of proteins from different groups of m and n sequences is defined as a number of mismatches in comparison of all possible m X n pairs of amino acid residues in the position (each from different group) divided by m X n. Ten position average of dissimilarity values is plotted vs. the first position number. Area of the figure between the profile of dissimilarity values and its mean value line characterizes the overall irregularity of amino acid substitutions along the protein sequences. If the area is greater than the average area for 1000 random profiles by more than two standard deviation units, the profile extrema containing the "surplus" of area are cut off. The cut-off stretches are likely to be variable and constant regions. If necessary, each of stretches may be separately tested and statistically estimated using a standard size sample of artificial protein families. Intergroup comparison of protein sequences reveals high overall irregularity of amino acid substitutions and identifies variable and conservative regions for all considered families of proteins: phospholipases A2, aspartate aminotransferases, alpha-subunits of Na+, K(+)-ATPase, L- and M-subunits of photosynthetic bacteria photoreaction centre, human rhodopsins.     

A microculture hybridization technique for the detection of specific DNA sequences in filamentous fungi

A procedure that permits the successful application of the technique of colony hybridization to the filamentous fungi is described. The method involves the growth of microcultures in the wells of a microtiter tray using spore inocula. These microcultures are protoplastedin situ and transferred to a nylon filter through a device made by drilling out the wells of a second microtiter tray. The transferred protoplasts are confined to defined areas of the membrane, where they may be lysed and subjected to hybridization with a radioactive probe. The technique gives highly reproducible results and is of sufficient sensitivity to detect the integration of a single copy of a transforming plasmid into the genome ofAspergillus nidulans.     

A contribution to breast cancer cell proteomics: detection of new sequences

Ductal infiltrating carcinoma (DIC) of the breast is the most common and potentially aggressive form of cancer. Knowledge of proteomic profiles, attained both in vivo and in vitro, is fundamental to acquire as much information as possible on the proteins expressed in these pathologic conditions. We used the breast cancer cell line 8701-BC, established from a primary DIC, with the aim of contributing to the databases on mammary cancer cells, which in turn will be very useful for the identification of differentially expressed proteins in normal and neoplastic cells. Within an analysis window comprising about 1750 discernible spots, we have at present catalogued 84 protein spots. The proteins for which an identity was assigned were identified essentially using gel comparison, N-terminal (Nt) microseqencing and immune detection. Among the protein spots Nt-microsequenced, sixteen corresponded to known proteins, four resulted as modified, relative to matching sequences deposited on databases, and seven were unknown. These modified or novel sequences are thus of potential interest to the knowledge of breast cancer proteomics and its applications.