New Perspectives on Unscheduled DNA Synthesis: Functional Assay for Global Genomic DNA Nucleotide Excision Repair.

The unscheduled DNA synthesis (UDS) assay measures the ability of a cell to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique by creating 6-4 photoproducts and pyrimidine dimers using UV-C (254 nm) irradiation. This procedure is designed specifically for quantification of the 6-4 photoproducts. Repair is quantified by the amount of radioactive thymidine incorporated during repair synthesis after this insult, and radioactivity is evaluated by grain counting after autoradiography. The results have been used to clinically diagnose human DNA repair deficiency disorders, and provide a basis for investigation of repair deficiency in human tissues or tumors. Genomic sequencing to establish the presence of specific mutations is also used now for clinical diagnosis of DNA repair deficiency syndromes. Few functional assays are available which directly measure the capacity to perform NER on the entire genome. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation (HCR). As discussed in Chap. 28 is not an equivalent technique, as it measures only transcription-coupled repair (TCR) at active genes, a small subset of total NER. Our laboratory also explored the fluorescent label-based Click-iT assay that uses EdU as the label, rather than 3H thymidine. Despite emerging studies in the literature finding this assay to be useful for other purposes, we found that the EdU-based UDS assay was not consistent or reproducible compared with the 3H thymidine-based assay.

Fe Foil-Guided Fabrication of Uniform Ag@AgX Nanowires for Sensitive Detection of Leukemia DNA.

Herein, we report a novel Fe foil-guided, in situ etching strategy for the preparation of highly uniform Ag@AgX (X = Cl, Br) nanowires (NWs) and applied the photoelectric-responsive materials for sensitive photoelectrochemical (PEC) detection of leukemia DNA. The Ag@AgX NW formation process was discussed from the redox potential and Ksp value. The fabricated PEC platform for sensing leukemia DNA showed good assay performance with a wide linear range (0.1 pM to 50 nM) and low detection limit of 0.033 pM. We envision that our Fe foil-guided synthetic method could be applied to synthesize more photoactive materials for sensitive PEC detections.

Urine RNA Processing in a Clinical Setting: Comparison of 3 Protocols.

OBJECTIVE: The objective of this study was to compare quantitative and qualitative RNA extraction results from clinical voided urine samples between 3 commercially available extraction protocols. METHODS: For phase 1, fresh voided urine samples from 10 female subjects were collected and processed in clinic and transported to the laboratory with cold packs. RNA was purified with 1 of 3 RNA extraction protocols: (1) TRI Reagent Protocol; (2) Absolutely RNA Nanoprep Kit; and (3) ZR Urine RNA Isolation Kit. Real-time polymerase chain reactions (RT-PCR) were performed. As the ZR Urine RNA Isolation Kit provided the highest quality RNA in phase 1, for phase 2, RNA was extracted from 9 additional voided urine specimens using this kit to perform additional qualitative analyses. RESULTS: Median RNA yield was significantly higher with the TRI Reagent Protocol as compared with the other protocols (P = 0.007). However, there was a significantly lower median threshold cycle value from polymerase chain reaction (indicating improved downstream application performance) with the ZR Urine RNA Isolation Kit as compared with the other methods (P = 0.005). In phase 2, the median RNA integrity number of urine RNA was 2.5 (range, 1.6-5.9). CONCLUSIONS: Although other methods may provide a higher quantity of RNA, when using clinical urine samples, the ZR Urine RNA Isolation Kit provided the highest quality of extracted RNA. This kit is especially attractive for the clinical setting because it does not require an initial centrifugation step. The urine RNA obtained with this kit may be useful for polymerase chain reaction but is not likely to be of high enough integrity for RNA sequencing.

Facile profiling of molecular heterogeneity by microfluidic digital melt.

This work presents a digital microfluidic platform called HYPER-Melt (high-density profiling and enumeration by melt) for highly parallelized copy-by-copy DNA molecular profiling. HYPER-Melt provides a facile means of detecting and assessing sequence variations of thousands of individual DNA molecules through digitization in a nanowell microchip array, allowing amplification and interrogation of individual template molecules by detecting HRM fluorescence changes due to sequence-dependent denaturation. As a model application, HYPER-Melt is used here for the detection and assessment of intermolecular heterogeneity of DNA methylation within the promoters of classical tumor suppressor genes. The capabilities of this platform are validated through serial dilutions of mixed epialleles, with demonstrated detection limits as low as 1 methylated variant in 2 million unmethylated templates (0.00005%) of a classic tumor suppressor gene, CDKN2A (p14ARF). The clinical potential of the platform is demonstrated using a digital assay for NDRG4, a tumor suppressor gene that is commonly methylated in colorectal cancer, in liquid biopsies of healthy and colorectal cancer patients. Overall, the platform provides the depth of information, simplicity of use, and single-molecule sensitivity necessary for rapid assessment of intermolecular variation contributing to genetic and epigenetic heterogeneity for challenging applications in embryogenesis, carcinogenesis, and rare biomarker detection.

A stainless-steel mortar, pestle and sleeve design for the efficient fragmentation of ancient bone.

Different types of milling equipment - such as oscillating ball mills, freezer mills, mortar and pestle - can be used to fragment ancient bone prior to DNA extraction. However, each of these tools is associated with practical drawbacks. Here, we present the design for a stainless-steel mortar and pestle, with a removable sleeve to contain bone material. The tool is easy to clean, practical and its simplicity allows university workshops equipped with a lathe, boring tools and a milling machine to make these components at local expense. This design allows for the efficient fragmentation of ancient bone and improves sample throughput. This design is recommended as a useful, economical addition to existing laboratory equipment for the handling of ancient bone.

A single quantum dot-based biosensor for DNA point mutation assay.

Sensitive and selective detection of point mutation is essential to molecular biology research and early clinical diagnosis. Here, we demonstrate a single quantum dot (QD)-based biosensor for DNA point mutation assay. In this assay, a mutant target (G/C) remains unchanged after the endonuclease treatment, and the polymerase chain reaction (PCR) may be initiated with the assistance of primers and polymerase, generating a large number of mutant targets. The amplified mutant targets can be captured by biotinylated probes during the process of denaturation and annealing, and Cy5-dGTP may be assembled into the biotinylated probe with the catalysis of polymerase, leading to the formation of Cy5-labeled biotinylated probes. The Cy5-labeled biotinylated probes can be further assembled onto the QD surface to obtain a Cy5-DNA-QD complex, resulting in the generation of fluorescence resonance energy transfer (FRET) between the QD donor and the Cy5 receptor. The mutant targets can be quantitatively evaluated by the measurement of Cy5 counts by total internal reflection fluorescence (TIRF) microscopy. While in the presence of wild-type targets (T/A), no Cy5-dGTP can be assembled into the biotinylated probe due to the presence of a mismatch and consequently no FRET is observed. This single QD-based biosensor exhibits high sensitivity with a detection limit of 5.3 aM (or 32 copies) and can even discriminate as low as 0.01% variant frequency from the mixture of mutant targets and wild-type ones. Importantly, this biosensor can be used for genomic analysis in human lung cancer cells, and may be further applied for an early clinical diagnosis and personalized medicine.

Remote-controlled release of DNA in living cells via simultaneous light and host-guest mediations.

Using photons as external triggers to realize remote-controlled release of oligonucleotide is superior to other intracellular or external stimulus. UV light is a valid photon-controlled manner due to high efficiency. However, further applications of these approaches in living cells are hampered by the large dose of UV-light irradiation. To address this issue, a simultaneous light and host/guest mediation was proposed in this paper. Gold nanoparticles (AuNPs) encoding with mercapto-ß-cyclodextrin (ßCD) served as a carried agent. Azobenzene (Azo), which was labeled on a releasing oligonucleotide, acted as a photochemically controlled switch. Ferrocene (Fc), an excellent guest for inclusion complexation by ßCD, serves as "enhancers" and shifts the equilibrium of the inclusion-exclusion process between trans-Azo and ßCD under UV-light irradiation, thus making the dose of UV-light irradiation reduced obviously. For further application, transfected green fluorescent protein (GFP)-expressing human lung cancer A549 cells were used to determine cellular uptake and gene silencing mediated by our constructed system in vivo. The results demonstrate that by employing Fc host-guest interaction, about 62.4% gene silencing was achieved within 30 min, which is significantly higher than that without Fc competition. Our strategy provides the potential for orthogonal DNA delivery and therapeutic activation that would be capable of achieving higher levels of site-specific activity and reduced amounts of side effects.

Kras gene codon 12 mutation detection enabled by gold nanoparticles conducted in a nanobioarray chip.

This study employs a nanobioarray (NBA) chip for multiple biodetection of single base pair mutations at the Kras gene codon 12. To distinguish between the mutant and wild-type target DNAs, current bioarray methods use high-temperature hybridization of the targets to the allele-specific probes. However, these techniques need prior temperature optimization and become harder to implement in the case of the detection of multiple mutations. We aimed to detect these mutations at a single temperature (room temperature), enabled by the use of gold nanoparticles (AuNPs) on the bioarray created within nanofluidic channels. In this method, a low amount of target oligonucleotides (5fmol) and polymerase chain reaction (PCR) products (300pg) were first loaded on the AuNP surface, and then these AuNP-bound targets were introduced into the channels of a polydimethylsiloxane (PDMS) glass chip. The targets hybridized to their complementary probes at the intersection of the target channels to the pre-printed oligonucleotide probe lines on the glass surface, creating a bioarray. Using this technique, fast and high-throughput multiple discrimination of the Kras gene codon 12 were achieved at room temperature using the NBA chip, and the specificity of the method was proved to be as high as that with the temperature stringency method.

Methylated-CpG Island Recovery Assay.

Alterations in DNA methylation patterns are implicated in playing a major role in the development of cancer, thus highlighting the need to continually develop new technologies to analyze epigenetic marks. Methylated-CpG Island Recovery Assay (MIRA), based on the high affinity of the MBD2b/MDB3L1 complex for double-stranded methylated DNA, allows for the recovery of methylated DNA without the use of bisulfite conversion or antibody recognition. MIRA is capable of detecting low-density methylation of a single methylated CpG nucleotide. This technique can be used in conjunction with microarrays or next-generation sequencing to analyze recovered methylated DNA on a genome-wide scale.

[Construction and selection of human Fab antibody phage display library of extracellular domain of HER 2].

AIM: To construct the fully humanized anti-extracellular domain (ECD) of HER2 Fab fragment phage library, select antibodies against HER2 ECD specifically and identify its characteristics. METHODS: Peripheral blood monouclear cells (PBMCs) of breast cancer patients with HER2-overexpressing were immunized in vitro with purification protein of recombinant HER2 ECD and were then transformed by Epstein-Barr virus (EBV). After total RNA was extracted, the heavy chain Fd and k/λ light chain were amplified by RT-PCR. Following restrictive digestion with Sac I/Xba I and Xho I/Spe I, the light chain k/λ genes and heavy chain genes Fd were inserted into the phagemid vector pComb3 successively and then electroporated into E.coil XL1-Blue.The humanized Fab phage antibody library against HER2 ECD was constructed by infection of helper phage VCSM13.The libraries were enrich after panned three cycles by purification protein of recombinant HER2 ECD.Then random clones were tested by ELISA to select the positive ones, which were furher identified their antigen binding acticities by Western blot, and the strongest binding to HER2 ECD clone was sequenced. RESULTS: The Fab phage antibody library with 2.5 x 10(7) volume was constructed and four positive clones which specifically recognized the HER2 ECD were isolated and further demonstrated by Western blot. Sequence analysis of the positivest clone showed that the variable heavy domains(VH) and variable light domains(VL) were highly homologous with the human embryonal Ig heavy chain V region sequences and kappa light chain sequences, respectively. CONCLUSION: A fully humanized Fab phage antibody library is successfully constructed and specific antibodies against HER2 ECD are obtained, which provides an experimental foundation for new humanized anti-HER2 ECD monoclonal antibodies.

Psychosocial issues in genetic testing for familial adenomatous polyposis: a review of the literature.

OBJECTIVES: Familial adenomatous polyposis (FAP) is characterized by the development of multiple adenomas in the colon that can lead to colorectal cancer. Being a carrier for FAP is hypothesized to have a negative impact on psychosocial well-being. This paper reviews the current literature on the psychosocial aspects of FAP. METHODS: Four literature databases were used to identify all papers published between 1986 and 2007 about psychosocial and behavioral issues in FAP related to genetic testing. The following topics were reviewed: uptake and psychosocial impact of genetic testing, endoscopic screening behavior and psychosocial well-being in general. RESULTS: Seventeen papers were identified. Across studies, genetic test uptake varied between 62 and 97%. Two out of three studies showed clinical levels of anxiety and/or depression after genetic testing. A minority of individuals were not reassured by a negative test result, and intended to continue endoscopic surveillance. Well-being (e.g. quality of life, family functioning) was found to be lower in some studies, while comparable to the general population in other studies. The studies had several shortcomings, such as mixed patient population (e.g. colorectal and breast cancer) and small sample sizes, and provided no information on other potentially important issues, such as psychosexual development. CONCLUSIONS: Future studies should employ larger sample sizes and standardized measurements. Additionally, future studies should address the long-term consequences of genetic testing for FAP, psychosexual development and consequences of FAP for the family as a whole.

Monitoring coping style moderates emotional reactions to genetic testing for hereditary nonpolyposis colorectal cancer: a longitudinal study.

OBJECTIVES: The emotional effects of genetic testing for hereditary nonpolyposis colorectal cancer (HNPCC) provided within a counseling program were assessed among 253 individuals. METHODS: Assessments were scheduled at baseline before testing, and again after 6 and 12 months post-test. Negative emotional reactions were evaluated using the Revised Impact of Event Scale and the Center for Epidemiological Studies-Depression Scale. Monitoring coping style was assessed at baseline using the Miller Behavioral Style Scale. RESULTS: Mean reductions were indicated in distress and depression levels within the first 6 months after counseling and testing. High monitors were generally more distressed than low monitors, specifically if they had indeterminate or positive results. CONCLUSIONS: Genetic counseling and testing for HNPCC do not result in long-term distress for most people. Of the variables investigated, only time and coping style have main effects on emotional reactions, and the impacts of mutation status are moderated by coping style. Psychological interventions, aimed to alleviate adverse emotional effects, were suggested for certain participants, i.e. recipients of positive or indeterminate results who are high monitors.

Molecular oncology: current trends in diagnostics.

Applications of molecular diagnostics to oncology have been slow to make their way to the clinical laboratory. While numerous genes and mutation spectra have been found to be involved in tumorigenesis, it is only recently that these findings begin to become useful in a clinical setting. Building on the technical knowledge obtained from molecular infectious disease testing, new instruments and assays have been developed to answer similar questions regarding qualitative, quantitative and genotyping issues. In this manuscript we describe two current examples of clinical molecular diagnostic applications, the assessment of BCR-ABL in chronic myelogenous leukemia patients and the detection of tumor cells in the sentinel lymph nodes of breast cancer patients, to demonstrate the role of molecular techniques in a routine clinical setting.

Prediction of microRNA targets.

Recently, microRNAs (miRNAs) have been shown to be important regulators of genes in many organisms and have already been implicated in a growing number of diseases. MiRNAs are short (21-23 nucleotides) RNAs that bind to the 3' untranslated regions of target genes. This binding event causes translational repression of the target gene and, evidence now suggests, also stimulates rapid degradation of the target transcript. miRNAs represent a new species of regulator, controlling the levels of potentially large numbers of proteins, many of which might be important drug targets. The expression of miRNAs shows that they are highly differentially expressed, with specific miRNAs active in certain tissues at certain times. In many cancers, miRNA expression is significantly altered, and this has been shown to be a useful diagnostic tool. Several computational approaches have been developed for the prediction of miRNA targets.

Hydroxyl radical footprinting in vivo: mapping macromolecular structures with synchrotron radiation.

We used a high flux synchrotron X-ray beam to map the structure of 16S rRNA and RNase P in viable bacteria in situ. A 300 ms exposure to the X-ray beam was sufficient for optimal cleavage of the phosphodiester backbone. The in vivo footprints of the 16S rRNA in frozen cells were similar to those obtained in vitro and were consistent with the predicted accessibility of the RNA backbone to hydroxyl radical. Protection or enhanced cleavage of certain nucleotides in vivo can be explained by interactions with tRNA and perturbation of the subunit interface. Thus, short exposures to a synchrotron X-ray beam can footprint the tertiary structure and protein contacts of RNA-protein complexes with nucleotide resolution in living cells.

[New developments in application of laser capture microdissection].

Isolation of pure targeted cells is an important and essential step for the molecular analysis of tissue lesion occurred in the progression of disease. Laser capture microdissection (LCM) is a novel technique that can be applied to obtain pure targeted cell subgroup or even a single cell quickly and precisely under the microscope, thus the problem of tissue heterogeneity in molecular analysis can be tackled successfully. In this article, the principles, advantages and disadvantages of LCM were introduced. New developments in the application of LCM were summarized in two fields (DNA analysis and gene expression analysis) respectively. Meanwhile, possible directions of the future developments of LCM were put forward.

The LightTyper: high-throughput genotyping using fluorescent melting curve analysis.

Instrumentation, chemistry, and software for high-throughput genotyping using fluorescent melting curves are described. The LightTyper system provides post-amplification genotyping within 10 min using samples in 96- or 384-well microplate formats. The system is homogenous because all reagents are added at the beginning of the reaction and there is no sample manipulation between amplification and genotyping. High-resolution melting curves are achieved by slow and steady heating. As samples are heated, panels of blue light-emitting diodes excite the probes, and fluorescence emission is acquired with a cooled charge-coupled device camera. A variety of probe chemistries are compatible for genotyping on the LightTyper, including dsDNA dyes, single-labeled probes, and fluorescence resonance energy transfer systems. Genotyping is performed automatically, and each sample is given a score reflecting the similarity of the genotype to the standards provided. Standard genotypes can be selected from within the run or imported from other files. Samples and genotypes can be grouped to allow multiple-allele detection on one or many samples. The utility of the LightTyper is illustrated by genotyping samples for the Factor V Leiden mutation and for mutations in the CFTR gene.

Single-nucleotide polymorphism analysis by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device.

We describe a microfluidic approach for allele-specific extension of fluorescently labeled nucleotides for scoring of single-nucleotide polymorphism (SNP). The method takes advantage of the fact that the reaction kinetics differs between matched and mismatched configurations of allele-specific primers hybridized to DNA template. A microfluidic flow-through device for biochemical reactions on beads was used to take advantage of the reaction kinetics to increase the sequence specificity of the DNA polymerase, discriminating mismatched configurations from matched. The volume of the reaction chamber was 12.5 nL. All three possible variants of an SNP site at codon 72 of the p53 gene were scored using our approach. This work demonstrates the possibility of scoring SNP by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device. The sensitive detection system and easy microfabrication of the microfluidic device enable further miniaturization and production of an array format of microfluidic devices for high-throughput SNP analysis.

[RNA isolation and purification methods]. / Les méthodes d'extraction et de purification des ARN.

Recent advances in human, bacterial and viral genome projects and the development of quantitative real-time reverse transcription-polymerase chain reaction methods offer the possibility of analysing a large number of gene transcripts. These molecular developments represent an important advancein the field of genetics, cancer, virology, bacteriology and hematology. A limiting step remains the isolation of high quality mRNA purified from biological samples. This review describes the different methods used to isolate mRNA from biological samples and to verify RNA integrity and gives precise details about RNA storage conditions.