Nathalie Zammatteo

New chips for molecular biology and diagnostics.

The DNA chips are arrays of DNA probes immobilized on solid support for simultaneous identification of many target DNA sequences. DNA chips applied to diagnosis aims to detect genomic DNA or RNA after PCR amplification. This review provides an overview of DNA chip technology, focusing on diagnostic applications. A comparison between high density and low density microarrays is given showing that low density chips are more suitable for routine applications due to their simplicity, good reproducibility, easy data management and low cost.

DNA probe hybridisation in microwells using a new bioluminescent system for the detection of PCR-amplified HIV-1 proviral DNA

A new bioluminescent detection system combined with a sandwich DNA hybridisation reaction in microwells has been developed for the detection of human immunodeficiency virus type 1 (HIV-1) provirus DNA amplified by the polymerase chain reaction (PCR). First, a fragment of the HIV-1 gag gene was amplified. The amplified DNA fragments were denatured and hybridised to a capture probe immobilised in microwells and to a biotinylated detection probe. A streptavidin-pyruvate kinase conjugate could then react on the biotinylated probe and the kinase activity detected by means of the luciferin-luciferase system, with production of a bioluminescent signal. This sandwich assay followed by a bioluminescent reaction detected as little as 7 amol of target DNA. The bioluminescent assay detected 5 HIV copies generated after one round of PCR, even if no band was seen on an agarose gel. The assay was applied to the detection of HIV-proviral DNA in peripheral blood mononuclear cells after one round of PCR and allowed to clearly identify a positive sample as compared to nested PCR.

A low-density DNA microarray for analysis of markers in breast cancer.

Breast cancer remains a major cause of death in women from Western countries. In the near future, advances in both nucleic acids technology and tumor biology should be widely exploited to improve the diagnosis, prognosis, and outcome prediction of this disease. The DNA microarray, also called biochip, is a promising tool for performing massive, simultaneous, fast, and standardized analyses of multiple molecular markers in tumor samples. However, most currently available microarrays are expensive, which is mainly due to the amount (several thousands) of different DNA capture sequences that they carry. While these high-density microarrays are best suited for basic studies, their introduction into the clinical routine remains hypothetical. We describe here the principles of a low-density microarray, carrying only a few hundreds of capture sequences specific to markers whose importance in breast cancer is generally recognized or suggested by the current medical literature. We provide a list of about 250 of these markers. We also examine some potential difficulties (homologies between marker and/or variant sequences, size of sequences, etc.) associated with the production of such a low-cost microarray.

Quantitative assay for group M (subtype A-H) and group O HIV-1 RNA detection in plasma.

A quantitative HIV-1 test is described based on a competitive RT-PCR assay combined with a sandwich hybridization as a detection system. The internal RNA standard (IS) was designed specifically to be competitive during the amplification and during the hybridization step. Sample viral load determination was carried out with one RT-PCR in the presence of 10(3) IS copies. The HIV-1 copy number was calculated by reference to an external standard curve performed on known and increasing amounts of the reference HIV-1 (Ref HIV-1) RNA co-amplified with a constant amount of the IS RNA. The assay had a linear range from 10(1) to 10(6) HIV-1 copies. HIV-1 strains belonging to the different subtypes from group M, but also group O, were all detected. Absolute quantification of purified HIV-1 RNA copies gave identical results as the AMPLICOR HIV-1 Monitor assay. The quantification of patients samples was evaluated according to different criteria such as dynamic range, sensitivity, efficacy of material recovery, reproducibility and convenience of sample handling. The microplate format of the assay combined with the colorimetric detection provides a convenient tool and fulfills the requirement for routine molecular diagnostic laboratories.

Comparison of three luminescent assays combined with a sandwich hybridization for the measurement of PCR-amplified human cytomegalovirus DNA

Identification of human tissue contaminated by cytomegalovirus is currently carried out by PCR amplification followed by measurement of the amplicons. Three luminescent detection systems undertaken after sandwich hybridization of the amplicons were compared. The sandwich hybridization takes place between a covalent linked capture probe, bound onto a plastic 96-well plate, and a biotinylated or digoxigenin-labeled detection probe. The three non-isotopic luminescent detection systems need either streptavidin-conjugated peroxydase or streptavidin-conjugated pyruvate kinase or antibodies conjugated with alkaline phosphatase. Detection of the enzymes was carried out by measurement of light emission in the presence of, respectively, luminol for peroxidase or dioxethane for alkaline phosphatase. The kinase assay was carried out not only in the presence of its substrates, ADP and phospho-enol pyruvate, but also of luciferase, which converts the produced ATP into light. The method was found to be sensitive, with the luciferase bioluminescent assay with the production of a long lasting signal. Amplicons from eight clinical samples were detected by this combination of sandwich hybridization and the three luminescent assays. The results were comparable with nested PCR for the identification of positive samples. The same correlation was obtained with 45 clinical samples using only the pyruvate kinase detection system. The high performance of these assays is given by the specificity of the sandwich hybridization combined with the sensitivity of the luminescent detection systems.