Detection of Non-Amplified Genomic DNA: 0 (Soft and Biological Matter)

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Harry Potter. Popular Features. New Releases. Description This book offers an overview of state-of-the-art in non amplified DNA detection methods and provides chemists, biochemists, biotechnologists and material scientists with an introduction to these methods. In fact all these fields have dedicated resources to the problem of nucleic acid detection, each contributing with their own specific methods and concepts.

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This book will explain the basic principles of the different non amplified DNA detection methods available, highlighting their respective advantages and limitations. Non-amplified DNA detection can be achieved by adopting different techniques. Such techniques have allowed the commercialization of innovative platforms for DNA detection that are expected to break into the DNA diagnostics market.

The enhanced sensitivity required for the detection of non amplified genomic DNA has prompted new strategies that can achieve ultrasensitivity by combining specific materials with specific detection tools. Advanced materials play multiple roles in ultrasensitive detection. Optical and electrochemical detection tools are among the most widely investigated to analyze non amplified nucleic acids. Biosensors based on piezoelectric crystal have been also used to detect unamplified genomic DNA. The main scientific topics related to DNA diagnostics are discussed by an outstanding set of authors with proven experience in this field.

Product details Format Paperback pages Dimensions x x Illustrations note 9 Tables, black and white; XIV, p. Other books in this series. Add to basket. The genome profile following Bst amplification was similar to the profile of the original sample. Solid line: expected 3 fold representational distortion. Dashed line: desired ratio of ideal WGA devoid of representational distortion. Comparison of array CGH genome profiles before and after Bst amplification.

NSCLC 8 2. NSCLC 9 2.

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Following, data was analyzed with aCGH-Smooth software, which identifies breakpoints and areas of gene amplification and deletion. The numbers presented refer to clones that were evaluable both before and after Bst amplification for each of the tumors. Lage and Dean et al [ 14 , 15 ] reported that MDA demonstrates high-amplification potential and excellent loci representation with less than 3-fold bias. We have demonstrated that in three groups of FFPE samples normal lung tissue, neuroblastoma xenografts and NSCLC , the relative content of different genes was maintained following the amplification.

If a bias existed, it was limited to a 3-fold change. We deliberately monitored the content of genes that are located on separate, unrelated regions of the genome to provide a good estimation of the overall amplification process. Hybridization against Bst -amplified reference DNA allows detection of genomic changes that are even below 3-fold change. This is more than the fold reported previously [ 14 ]. The discrepancy might be attributed to the method used for quantitation of the template and products PicoGreen DNA quantitation vs.

NanoDrop spectrophotometry. The wide range in amplification yield may be due to variability in DNA quality and tissue fixation. However, the partial shearing of intact genomic DNA did not result in a significant change of the amplification yield. Yet, our results contradict previous mathematically-based predictions of a lower yield with sheared DNA [ 14 ]. Our QPCR analysis of gene copy content of FFPE samples following Bst amplification demonstrated up to a 3-fold change with respect to the non-amplified samples, fitting earlier reports of up to 3-fold representational bias [ 14 , 15 ].

To compare the bias resulting from various WGA methods is challenging, as the reported characteristics of each method depend on the initial amount of DNA template used, as well as on the application under investigation. The representational bias can be implied from the reported range of efficiency rates for amplification of DNA sequences of several microsatellite loci.

References

This is thought to result from spurious amplification of the primers. Lage et al [ 14 ] reported that background DNA synthesis was completely suppressed when modified primers with two 5'-nitroindole universal base residues were used. We were unable to eliminate primer amplification reactions despite the use of modified primers.


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However, this spurious primer amplification appears to be tolerable, since tested genes were consistently detected by QPCR in all the Bst -amplified samples but not in the amplified negative control. In contrast, Bst DNA polymerase can switch templates and is therefore less affected by the proximity of genes to the telomere [ 14 ]. Copy number changes detected following Bst amplification are reliable only if they are higher than the 3-fold representational distortion range. Thus, it is expected that high copy number changes e. N- myc in neuroblastoma xenografts will be easily detected, since even with 3-fold amplification bias, the change in copy number is conspicuous compared to normal gene content.

Although low copy number changes e. Skp2 in NSCLC are also detectable, the difference in copy number relative to normal gene content may be attenuated with 3-fold representation distortion. We found concordance of Lage stated that altered loci with relatively high gene-dosage alterations were detected with high reproducibility. Likewise, we observed that gene amplifications were consistently detected, even when smaller than 3-fold, yet the detection sensitivity correlated with the level of genomic changes.

We noted that gene deletions were prone to be missed following Bst amplification and similar findings can be seen in the data presented by Lage et al. Array properties, the quality of the amplified DNA, the length of deleted areas and the low level of gene content change in deletions, which is strongly affected by the amplification representation bias introduced by MDA, all contribute to the reduced detection ability of deletions following Bst amplification.

However, there has recently been a growing interest in the method, as reflected by the increasing number of papers published during the past two years. MDA was reported to succeed in a variety of applications, including sequencing [ 20 , 21 ], microsatellite marker analysis [ 22 , 23 ], SNP analysis [ 24 , 25 ], genotyping [ 26 ] and array-CGH [ 14 , 27 ]. Although the reaction yielded DNA that was fold greater than the initial amount, no genes could be detected by QPCR and results were consistent with a spurious amplification of the primers. However, this technique requires purification following each step and is therefore laborious and vulnerable to sample loss [ 19 , 29 ].

The expected application, magnitude of findings and the limits of the method are factors that need to be considered when WGA is chosen. We believe that in time, Bst DNA amplification will become part of routine molecular laboratory work for research and clinical purposes. Snap-frozen tissues were banked within 30 min after resection. Seven non-small cell lung cancer NSCLC samples with known Skp2 gene amplification [ 37 ] were also similarly evaluated. Tumor cells from the xenograft and NSCLC samples were enriched by manual microdissection from sections stained by toluidine blue.

DNA was sheared by sonication to strands of less than 4 Kb. Digestion with 0.


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  7. Briefly, 10 ng of DNA were mixed with 1. By combining two tests evaluating different ALT characteristics in this protocol, the problem of equivocal results can also be minimized. Assessing embryonal tumors such as NB for ALT by TRF or TC can be particularly challenging as the separation between the two groups is often not as distinct as in adult tumors, possibly because less telomere attrition occurs during the genesis of embryonal tumors. Evaluating two characteristics simultaneously also increases both the sensitivity and specificity for ALT detection. However, selecting SCG in cancer cells can be challenging as gene copy number variations are common in cancer cells and there are significant differences in affected chromosome regions among different cancers.

    Fortunately, comparative genomic hybridization array data are now widely available for most tumor types and can direct the selection of SCG. Care must also be taken when evaluating the quantity of SCG product in each sample to ensure the results are consistent with the behavior of a SCG. Furthermore, qPCR requires normalization to a reference sample. In the setting of this assay, the cut-offs can only be applied when normalization is accurate.

    We therefore provided relative qPCR measurements obtained from three commercially available ALT cell lines at specified passage.

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    This allows the user of this qPCR-based assay to perform multi-point calibration for both TC and CC measurements prior to testing unknown samples and to ensure that the cut-off values, normalized to U-2 OS, can be accurately applied. In addition to research applications, this ALT detection protocol can be readily adopted by clinical laboratories.

    Many clinical laboratories routinely perform qPCR now, but are not equipped for Southern-blot analysis and for techniques involving radiation. The ability to diagnose ALT efficiently in both research and clinical settings will be crucial for the development of ALT inhibitors as anticancer therapies and for choosing the appropriate telomere maintenance mechanism-targeted drugs in the clinic.

    In conclusion, this novel protocol is the first to detect ALT by telomere qPCR and is feasible in clinical pathology laboratories. This radio-isotope-free assay has advantages over the original CC assay 8 , which requires more DNA for the detection of one characteristic CC , uses radioactive probe and is more labor-intensive. The concurrent evaluation of two distinct ALT characteristics increases the sensitivity and specificity of ALT detection and the format of the assay together with its requirement for only a small amount of DNA will facilitate medium-throughput screening of large tumor cohorts for ALT.

    Funding for open access charge: Children's Medical Research Institute. Conflict of interest statement. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Advanced Search. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents.

    Oxford Academic. Google Scholar. Rebecca A. Jeremy D. Amy Y. Janice A. Roger R. Article history. Revision Received:. Cite Citation. Permissions Icon Permissions. Abstract Alternative lengthening of telomeres ALT is one of the two known telomere length maintenance mechanisms that are essential for the unlimited proliferation potential of cancer cells.

    Figure 1. View large Download slide. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Search ADS. Assaying and investigating Alternative Lengthening of Telomeres activity in human cells and cancers. A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas.

    Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia PML body. Prevalence of the alternative lengthening of telomeres telomere maintenance mechanism in human cancer subtypes. DNA C-circles are specific and quantifiable markers of alternative-lengthening-of-telomeres activity. Association of marine omega-3 fatty acid levels with telomeric aging in patients with coronary heart disease.

    Telomere length in human adults and high level natural background radiation. Array comparative genomic hybridization identifies genetic subgroups in grade 4 human astrocytoma.



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