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      Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

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          Abstract

          Background

          Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive.

          Results

          We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy.

          Conclusion

          Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

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          Most cited references 33

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          Changes in thymic function with age and during the treatment of HIV infection.

          The thymus represents the major site of the production and generation of T cells expressing alphabeta-type T-cell antigen receptors. Age-related involution may affect the ability of the thymus to reconstitute T cells expressing CD4 cell-surface antigens that are lost during HIV infection; this effect has been seen after chemotherapy and bone-marrow transplantation. Adult HIV-infected patients treated with highly active antiretroviral therapy (HAART) show a progressive increase in their number of naive CD4-positive T cells. These cells could arise through expansion of existing naive T cells in the periphery or through thymic production of new naive T cells. Here we quantify thymic output by measuring the excisional DNA products of TCR-gene rearrangement. We find that, although thymic function declines with age, substantial output is maintained into late adulthood. HIV infection leads to a decrease in thymic function that can be measured in the peripheral blood and lymphoid tissues. In adults treated with HAART, there is a rapid and sustained increase in thymic output in most subjects. These results indicate that the adult thymus can contribute to immune reconstitution following HAART.
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            Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase.

            The 5'----3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification. An oligonucleotide probe, nonextendable at the 3' end, labeled at the 5' end, and designed to hybridize within the target sequence, is introduced into the polymerase chain reaction assay. Annealing of probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5'----3' exonuclease activity of T. aquaticus DNA polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe. The assay is sensitive and specific and is a significant improvement over more cumbersome detection methods.
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              T cell homeostasis in patients with rheumatoid arthritis.

              The immune system is equipped with an extremely large spectrum of structurally diverse receptors to recognize all potential antigens. This fundamental principle of receptor diversity is no longer upheld in patients with rheumatoid arthritis (RA), who have a marked contraction of the T cell receptor repertoire. In this study, the ability of RA patients to produce T cells and to maintain T cell homeostasis was examined. CD4 T cells containing T cell receptor rearrangement excision circles (TREC) were substantially reduced in RA patients; TREC levels in young adult patients matched those of controls 20 years older. Increased self-replication of T cells in RA was indicated by age-inappropriate erosion of telomeres in circulating T cells with almost complete attrition of telomeric reserves in patients 20-30 yr of age. The degree of telomere loss was not related to disease duration or the use of disease-modifying medication and was most pronounced in CD4(+)CD45RO(null) (naive) T cells. The loss of TREC-positive T cells could be a consequence of a primary defect in peripheral T cell homeostasis. Alternatively, RA patients may have impaired thymic function with the increased turnover of peripheral T cells being a secondary compensatory event.
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                Author and article information

                Journal
                BMC Biotechnol
                BMC Biotechnology
                BioMed Central (London )
                1472-6750
                2003
                13 October 2003
                : 3
                : 18
                Affiliations
                [1 ]Molecular Medicine Unit, University of Leeds, Leeds, UK
                [2 ]Centre Anti-cancer Leon Berard, Lyon, France
                Article
                1472-6750-3-18
                10.1186/1472-6750-3-18
                270040
                14552656
                Copyright © 2003 Ponchel et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.
                Categories
                Methodology Article

                Biotechnology

                amplification, real-time pcr, rearrangement, sybr-green, deletion

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