A rapid, cost-effective method of assembly and purification of synthetic DNA probes >100 bp.

Here we introduce a rapid, cost-effective method of generating molecular DNA probes in just under 15 minutes without the need for expensive, time-consuming gel-extraction steps. As an example, we enzymatically concatenated six variable strands (50 bp) with a common strand sequence (51 bp) in a single pool using Fast-Link DNA ligase to produce 101 bp targets (10 min). Unincorporated species were then filtered out by passing the crude reaction through a size-exclusion column (<5 min). We then compared full-length product yield of crude and purified samples using HPLC analysis; the results of which clearly show our method yields three-quarters that of the crude sample (50% higher than by gel-extraction). And while we substantially reduced the amount of unligated product with our filtration process, higher purity and yield, with an increase in number of stands per reaction (>12) could be achieved with further optimization. Moreover, for large-scale assays, we envision this method to be fully automated with the use of robotics such as the Biomek FX; here, potentially thousands of samples could be pooled, ligated and purified in either a 96, 384 or 1536-well platform in just minutes.

Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis.

The ability to visualize specific DNA sequences, on chromosomes and in nuclei, by fluorescence in situ hybridization (FISH) is fundamental to many aspects of genetics, genomics and cell biology. Probe selection is currently limited by the availability of DNA clones or the appropriate pool of DNA sequences for PCR amplification. Here, we show that liquid-phase probe pools from sequence capture technology can be adapted to generate fluorescently labelled pools of oligonucleotides that are very effective as repeat-free FISH probes in mammalian cells. As well as detection of small (15 kb) and larger (100 kb) specific loci in both cultured cells and tissue sections, we show that complex oligonucleotide pools can be used as probes to visualize features of nuclear organization. Using this approach, we dramatically reveal the disposition of exons around the outside of a chromosome territory core and away from the nuclear periphery.

Molecular cytogenetic methodologies and a BAC probe panel resource for genomic analyses in the zebrafish.

Molecular cytogenetics is a field that emerged in the 1980s, based on a technique referred to as fluorescence in situ hybridization, (FISH). Using FISH methodologies, a specific DNA sequence or collection of DNA fragments may be selectively labeled with a hapten molecule or fluorescent dye and hybridized to denatured chromosomes, interphase cells, or even chromatin fibers. DNA hybridization kinetics permit these labeled probes to anneal to their complementary sequences on such chromosomal DNA preparations allowing for direct visualization of the sequence of interest in the genome being interrogated. If present, the relative chromosomal position of the sequence can sometimes also be ascertained. Progress in molecular cytogenetic research has advanced the genetic characterization of zebrafish models of human diseases as well as assisted with accurate annotation of the zebrafish reference genome by anchoring large DNA fragments to specific chromosome regions. Using the procedures described in this chapter, hundreds of ambiguous zebrafish bacterial artificial chromosome (BAC) clones have already been assigned to individual genetic linkage groups. Molecular cytogenetic techniques can also be used to study gene duplication events and study the molecular mechanisms by which they arise. Moreover, the availability of a new molecular cytogenetic technique, array-based comparative genomic hybridization (aCGH), is now able to identify gains and losses of DNA segments in zebrafish DNA samples in a genome-wide manner and in a single assay.

Generation of an enriched pool of DNA aptamers for an HER2-overexpressing cell line selected by Cell SELEX.

Overexpression of human epidermal growth factor receptor 2 (HER2) occurs in a large percentage of breast cancers. Monoclonal antibodies targeting HER2 are vastly used for both diagnostic and therapeutic aims. However, identifying a new molecular probe against HER2 with improved diagnostic and therapeutic features is of great importance. In this report, we have applied the cell systematic evolution of ligands by exponential enrichment (SELEX) strategy for 16 selection rounds to generate an enriched pool of aptamers that specifically recognize the HER2 positive cell line. During the Cell SELEX procedure, a human HER2-overexpressing breast cancer cell line and a human HER2 negative breast cancer cell line were used. Our results reveal that polymerase chain reaction (PCR) amplification of random DNA libraries and the selected single-stranded DNA pool in different Cell SELEX rounds are different from what we expect from PCR amplification of homologous DNA. Our results also confirmed previous studies describing positive HER2 status of SK-BR3 and the absence of the HER2 expression in the MDA-MB468. We also developed a new method, Cell enzyme-linked assay, to monitor the enrichment of aptamers in a given round of Cell SELEX. This method would also be useful in other experiments using live cell enzyme-linked immunosorbent assay on adherent cells.

Copy number variation in the complement factor H-related genes and age-related macular degeneration.

PURPOSE: To determine the contribution of copy number variation (CNV) in the regulation of complement activation (RCA) locus to the development of age-related macular degeneration (AMD). METHODS: A multiplex ligation-dependent probe amplification assay was developed to quantify the number of copies of CFH, CFHR3, CFHR1, CFHR4, CFHR2, and CFHR5 in humans. Subjects with (451) and without (362) AMD were genotyped using the assay, and the impact on AMD risk was evaluated. RESULTS: Eight unique combinations of copy number variation were observed in the 813 subjects. Combined deletion of CFHR3 and CFHR1 was protective (OR=0.47, 95% confidence interval 0.36-0.62) against AMD and was observed in 88 (82 [18.6%] with one deletion, 6 [1.4%] with two deletions) subjects with AMD and 127 (108 [30.7%] with one deletion, 19 [5.4%] with two deletions) subjects without AMD. Other deletions were much less common: CFH intron 1 (n=2), CFH exon 18 (n=2), combined CFH exon 18 and CFHR3 (n=1), CFHR3 (n=2), CFHR1 (n=1), combined CFHR1 and CFHR4 (n=15), and CFHR2 deletion (n=7, 0.9%). The combined CFHR3 and CFHR1 deletion was observed on a common protective haplotype, while the others appeared to have arisen on multiple different haplotypes. CONCLUSIONS: We found copy number variations of CFHR3, CFHR1, CFHR4, and CFHR2. Combined deletion of CFHR3 and CFHR1 was associated with a decreased risk of developing AMD. Other deletions were not sufficiently common to have a statistically detectable impact on the risk of AMD, and duplications were not observed.

Laser-capture microdissection of developing barley seeds and cDNA array analysis of selected tissues.

Laser microdissection provides a useful method for isolating specific cell types from complex biological samples for downstream applications. In contrast to the texture of mammalian cells, most plant tissues exhibit a cell organization with hard, cellulose-containing cell walls, large vacuoles, and air spaces, thus complicating tissue preparation and extraction of macromolecules such as DNA and RNA. Especially, barley seeds show cell types with enormous differences in osmolarity (degenerating and differentiating tissues) and contain high amounts of the main storage product starch, thus requiring specific procedures for morphological preservation and RNA extraction. In this study, we report about methods allowing tissue-specific gene expression profiling of developing barley seeds. Details on aspects of tissue preparation, including fixation and embedding procedures, laser-capture microdissection, RNA isolation, and linear mRNA amplification to produce high-quality labelled probes for large-scale expression analysis are provided. Particular emphasis is placed on the fidelity of transcript data obtained by the developed methods in relation to the in vivo transcriptome.

A real-time Taqman method for hepatitis C virus genotyping and methods for further subtyping of isolates.

The HCV genome is highly heterogeneous; more and more genotypes, each with several distinct subtypes, are being identified around the world. Knowledge of genotype is important for planning of treatment regimes, whereas subtype identification is useful in epidemiological studies and outbreak investigation. We describe HCV genotyping and subtyping assays, based on real-time PCR, that are sensitive, specific, and reliable. These assays provide fast, accurate, and convenient methods for HCV genotyping/subtyping to support clinical practice.

Probe production for in situ hybridization by PCR and subsequent covalent labeling with fluorescent dyes.

A simple procedure for fluorescent labeling of probes just before in situ hybridization is provided. Aminoallyl-dUTP is introduced during probe production by polymerase chain reaction (PCR). The aminoallyl-dUTP functions as a reactive site for subsequent labeling of the probe. Activated fluorescent dyes such as fluorescein are covalently attached to the probe through the formation of a stable amide bond. Labeled probes are purified by size-exclusion gel chromatography to remove unincorporated dye. Target genes used to demonstrate the efficacy of this technique with in situ hybridization are rat Y-chromosome and rat granulocyte colony-stimulating factor receptor. PCR amplicons containing aminoallyl-dUTP were produced in high yield. Probes obtained after labeling with activated fluorophores demonstrated high intrinsic activity within in situ hybridizations. The introduction of aminoallyl-dUTP into the PCR reaction enables the production of "unlabeled" probes by PCR having a shelf life, which is not limited by the storage and stability challenges of fluorophore-labeled probes. Subsequent labeling of the probes with activated fluorescent dyes just before use allows one step in situ hybridization with high activity and minimal background staining.

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization.

Chromogenic (CISH) and fluorescent (FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes (BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12;15)(p12;q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.

Design and preparation of Epstein-Barr virus genome-wide cDNA probes.

OBJECTIVE: To design and clone all known and predicted coding genes of Epstein-Barr virus (EBV) as the cDNA probes for preparing the microarray for EBV detection, thereby to facilitate further investigation of the pathogenetic role of EBV. METHODS: Oligo 6.0 software, BLAST program and Primer Premier 5 software were employed to design and screen the cDNA probes of the whole EBV genome, whose length ranged from 300 to 600 mer each with high specificity. These cDNA probes obtained through PCR and reverse transcriptase (RT)-PCR amplification from the genomic DNA and RNA of B95-8 cells and nasopharyngeal carcinoma (NPC) tissue were cloned into T/A clone vector, followed by identification of these probes by sequencing analysis. RESULT AND CONCLUSION: A total of 85 gene fragments (BWRF1 gene-contained 7 repeats of open reading frames) coding for proteins and 2 EBERs in EBV genome were successfully cloned, not including LF1 and LF3 genes that did not exist in EBV genome of B95-8 cells, which provides the basis for preparing microarray to explore the role of EBV genome in its related diseases.

FlyGEM, a full transcriptome array platform for the Drosophila community.

We have constructed a DNA microarray to monitor expression of predicted genes in Drosophila. By using homotypic hybridizations, we show that the array performs reproducibly, that dye effects are minimal, and that array results agree with systematic northern blotting. The array gene list has been extensively annotated and linked-out to other databases. Incyte and the NIH have made the platform available to the community via academic microarray facilities selected by an NIH committee.

Probe generation directly from small numbers of cells for DNA microarray studies.

Recently, we described a technique that allows us to prepare probes for expression profiling from 0.5-1 microgram RNA without template or signal amplification. However, we were unable to use this method to study cells harvested by needle biopsy, cell sorting, or laser capture microdissection. Here we give a new protocol for amplifying RNA with multiple reaction cycles and preparing fluorescent probes from approximately 10 cells. We use random 9-mers with a T3 RNA polymerase recognition sequence on the 5' end for every round of cDNA synthesis except the first. The latter is primed with oligo(dT) with a T7 RNA polymerase recognition sequence on the 5' end. Results were highly reproducible and reliable, and the products generated using our method seemed comparable to those produced using the RiboAmp RNA kit when both were used to do two cycles of amplification. To test our method's utility, we lysed cells directly into reverse transcription buffer containing RNase inhibitor and performed three rounds of RNA amplification. The expression profiles of mouse C2 and NIH 3T3 cells obtained with 11,232-element arrays using amplified RNAs were similar to those seen when probes were prepared from unamplified templates.

Automated high-throughput probe production for DNA microarray analysis.

DNA microarrays have become an established tool for gene expression profiling. Construction of these microarrays using immobilized cDNAs is a common experimental strategy. However, this is extremely laborious, requiring the preparation of hundreds or thousands of cDNA probes. To minimize this initial bottleneck, we developed a comprehensive high-throughput robotic system to prepare DNA probes suitable for microarray analysis with minimal user intervention. We describe an automated system using the MultiPROBE Nucleic Acid Purification Workstation to provide the liquid handling and other functions needed to optimize this process. We were able to carry out fully automated plasmid cDNA isolation, PCR assay setup, and PCR purification and also to direct the characterization and tracking of DNA probes during processing. Protocols began with the initial preparation of a plasmid DNA archive of bacterial stocks in parallel 96-well plates (192 samples/run) and continued through to the dilution and reformatting of chip-ready DNA probes in 384-well format. These and other probe production procedures and additional instrument systems were used to process fully a set of mouse cDNA clones that were then validated by differential gene expression analysis.

RNA analysis by nuclease protection.

Nuclease protection assays (S1 nuclease protection and RNase protection) are extremely sensitive procedures for detection and quantitation of mRNA species in complex mixtures of total cellular RNA. These assays are well suited for mapping positions of external and internal junctions in RNA, such as transcription initiation and termination sites and intron/exon boundaries, and to discriminate between closely related targets by using probes designed to span the regions where the related genes differ the most. Also, because the size of the probes used in nuclease protection assays is a variable chosen by the investigator, probes may be designed to protect fragments of different sizes. This feature permits the simultaneous analysis of several different mRNAs in the same total RNA sample. In this unit, a method is included for RNase protection of target mRNA sequences, including hybridization of the probe to the target sequence, details of the actual protection assay, and detection of reaction products. An alternative method is provided for performing the RNase protection assay on a microvolume scale, which is useful when there are many samples to be analyzed. Support protocols describe synthesis and gel purification of labeled RNA probes; preparation of RNase-free yeast RNA, which acts as an aid in the quantitative precipitation of newly synthesized probe; and quantitation of target mRNA. A method describing S1 nuclease protection of target mRNA using either RNA or DNA probes is also included. Additional support protocols provide instructions for the preparation of radiolabeled DNA probes by primer-extension of double-stranded plasmid or PCR product using Klenow fragment of E. coli DNA polymerase I or Taq or Tth polymerase in a thermal cycler. Another radiolabeling method details 5' end labeling of oligodeoxynucleotides and oligoribonucleotides using T4 polynucleotide kinase. Additionally, a method is described for mapping transcription start sites using the S1 nuclease protection assay.

Seryl-histidine as an alternative DNA nicking agent in nick translation yields superior DNA probes and hybridizations.

Nick translation is a commonly used method for labeling DNA to make DNA hybridization probes. In this approach, the use of DNase I to generate nicks in double-stranded DNA presents an inherent drawback, because the enzyme's high rate of reaction causes significant fragmentation and shortening of the hybridization probes. Based on our recent findings regarding the nucleolytic activity of the dipeptide seryl-histidine (Ser-His) and generation of free 3' hydroxyl and 5' phosphate groups at the cleavage sites of the substrate DNA by Ser-His, it was hypothesized that this disadvantage may be overcome by using Ser-His in place of DNase I as an alternative DNA nicking agent. In this study we demonstrate that like DNase I, Ser-His randomly nicks DNA, but the dipeptide has a much lower rate of reaction that enables more complete labeling of the DNA probes with less fragmentation. DNA probes labeled through nick translation using Ser-His as the DNA nicking agent were consistently larger in size and exhibited significantly higher specific activities, and enhanced hybridization signals in Southern blot analyses compared to control DNA probes that were made using DNase I as the nicking agent. Furthermore, the degree of nicking and consequently the quality of the probes could be easily controlled by adjusting the temperature and time of the Ser-His nicking reaction. These results affirm our hypothesis that Ser-His can serve as an alternative DNA nicking agent in nick translation to yield superior DNA probes and hybridization results and suggest the possible general utility of Ser-His for wide range of biological and biomedical applications that require more moderated nicking of nucleic acids. Based upon these and computer modeling results of Ser-His, a mechanism of action is proposed to explain how Ser-His may nick DNA.

An evaluation of the performance of cDNA microarrays for detecting changes in global mRNA expression.

The cDNA microarray is one technological approach that has the potential to accurately measure changes in global mRNA expression levels. We report an assessment of an optimized cDNA microarray platform to generate accurate, precise and reliable data consistent with the objective of using microarrays as an acquisition platform to populate gene expression databases. The study design consisted of two independent evaluations with 70 arrays from two different manufactured lots and used three human tissue sources as samples: placenta, brain and heart. Overall signal response was linear over three orders of magnitude and the sensitivity for any element was estimated to be 2 pg mRNA. The calculated coefficient of variation for differential expression for all non-differentiated elements was 12-14% across the entire signal range and did not vary with array batch or tissue source. The minimum detectable fold change for differential expression was 1.4. Accuracy, in terms of bias (observed minus expected differential expression ratio), was less than 1 part in 10 000 for all non-differentiated elements. The results presented in this report demonstrate the reproducible performance of the cDNA microarray technology platform and the methods provide a useful framework for evaluating other technologies that monitor changes in global mRNA expression.

Enzymatic labeling of nucleic acids.

Extensive knowledge of the enzymology involved in biosynthesis and degradation of nucleic acids has permitted the development of simple methods for labeling RNA and DNA with radioisotopes or biotin. These labeled probes are used primarily for hybridization to nucleic acid fragments of interest in a variety of applications. A complete description of the methods available for such labeling is beyond the scope of this manual, but contained within this unit are protocols for oligonucleotide-primed synthesis of radiolabeled and biotinylated DNA probes, in vitro synthesis of radiolabeled RNA, and end labeling of synthetic oligonucleotide probes.

[The preparation of norA gene probe in Staphylococcus aureus].

OBJECTIVE: To prepare norA gene probe for studies on the mechanism of resistance to fluoroquinolones mediated by norA gene in S. aureus. METHODS: The Dig-labeled norA gene probe was made by PCR. RESULTS: This method is simple and feasible. We have got a lot of probes in a short time. The sensitivity of norA gene probe is high; the probe is secure, easy to use and can be stored for a long time. CONCLUSION: The probe is applicable to further studies on the mechanism of resistance to fluoroquinolones mediated by norA in S. aureus.