Compositions and methods for detecting a sequence mutation...

C - Chemistry – Metallurgy – 12 – Q

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C12Q 1/68 (2006.01) G01N 33/46 (2006.01)

Patent

CA 2515211

Loblolly pine (Pinus taeda L.) is the most important commercial tree species in the USA harvested for pulp and solid wood products. Increasing the efficiency of chemical pulping may be achieved through the manipulation of genes involved in the lignin biosynthetic pathway. A null allele of cinnamyl alcohol dehydrogenase (CAD) has been discovered in the loblolly pine clone 7- 56 which displays altered lignin composition. During identification of single nucleotide polymorphisms (SNPs) in the cad gene, a two-base pair adenosine insertion located in exon five and unique to clone 7-56 was discovered. The sequence mutation causes a frame-shift predicted to result in premature termination of the protein. For routine detection of the mutation, a diagnostic assay was developed utilizing Template-directed Dye-terminator Incorporation and Fluorescence Polarization detection (FT-TDI).

Aux Etats-Unis, le pin taeda (Pinus taeda L.) est la principale espèce d'arbre commercial récoltée pour la fabrication de pâte et de produits en bois massif. La manipulation de gènes impliqués dans la voie biosynthétique de la lignine permet d'obtenir une augmentation de l'efficacité de réduction en pâte chimique. On a découvert un allèle nul de l'alcool cinnamylique déshydrogénase (CAD) dans le clone du pin taeda 7-56 présentant une composition de lignine modifiée. Lors de l'identification de polymorphismes mononucléotidiques (SNP) dans le gène cad, on a découvert une insertion d'adénosine à deux paires de bases située dans l'exon cinq et unique pour le clone 7-56. La mutation de séquence provoque un changement de phase impliquant une terminaison prématurée de la protéine. Pour une détection de routine de la mutation, on a développé une méthode diagnostique faisant appel à une technique d'immunodosage dirigé par matrice à colorant-terminateur avec polarisation de fluorescence (FP-TDI).

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