Utilization of nucleotide probes for the measurement of...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

Reexamination Certificate

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C435S091100, C435S091200, C536S023100, C536S024300, C536S026600

Reexamination Certificate

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06924102

ABSTRACT:
The present invention concerns the development of a quantitative method for the molecular diagnosis of autosomal recessive spinal muscular atrophy (SMA) by measuring the amount of cytosolic mRNA from human muscle cells. Both the procedure using radioactive material and the Enzyme-Linked Immunosorbent Assay (ELISA) nonradioactive method were developed using32P-dCTP labeled and biotinylated nucleotide probes. The results obtained demonstrate that the measurement of mRNA could be used as a quantitative method for the molecular diagnosis of SMA. There was a perfect concordance of the results obtained between the procedure using radioactive material, the ELISA method and the single strand conformation polymorphism (SSCP) analysis regarding the negative and positive SMA samples. The methods developed in this study may be applicable to the diagnosis (detection of homozygous and heterozygous deletions in exons 7 and 8 of the SMN gene) and the control of mRNA concentrations in the future gene therapy of patients with SMA.

REFERENCES:
patent: 5635351 (1997-06-01), Feuerstein et al.
Jong et al. “Analysis of the mRNA transcripts of the SMN gene in the tissue of an SMA fetus and the peripheral blood mononuclear cells of normals, carriers and SMA patients.” J. of the Neurological Sciences. vol. 173, pp. 147-153, Feb. 2000.
Gruber et al. (Histochem Cell Biol. vol. 107, pp. 411-416, 1997).
Bruce (Am J. Respir. Cell Mol. Biol. vol. 20, pp. 228-236, 1999).
Van Der Steege et al. “PCR-based DNA test to confirm clinical diagnosis of autosomal recessive spinal muscular atrophy.” Lancet. vol. 345, pp. 985-986, Apr. 1995.
Lefebvre et al. (Genbank Accession No. U438836, May 16, 1996).
Lefebvre et al. “Identification and Characterization of a spinal muscular atrophy-determining gene.” Cell, vol. 80, pp. 155-165, Jan. 13, 1995.

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