C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
C12N 15/10 (2006.01) C07H 21/00 (2006.01) C12P 19/34 (2006.01) C12Q 1/68 (2006.01) C40B 30/00 (2006.01)
Patent
CA 2256606
Improved methods for obtaining polynucleotides comprising sequences which differ between two populations of DNA or cDNA are provided. Improvements include reduction in the number of amplification cycles, use of a nuclease digestion step prior to amplification, a novel oligonucleotide adapter for the practice of the improved method, and novel methods for selective amplification of the desired unique fragments and selective degradation of fragments containing sequences common to both populations. Fragments of a sample population are amplified using a primer that endows the amplification products with resistance to nuclease degradation. Fragments of a control population are amplified using a primer that targets the amplification products for preferential degradation. Multiple cycles of hybridization, nuclease treatment and amplification are utilized to provide enrichment of fragments unique to the sample population. Such unique fragments may represent new or amplified sequences in the sample population, sequences that are differently arranged in the sample population compared to the control population, and sequences that are differentially expressed in a cDNA population. A variation of the technique also allows the isolation of fragments representing deletions in the sample population.
Cette invention constitue un perfectionnement d'un procédé d'obtention de polynucléotides dont des séquences se différencient d'une population d'ADN ou d'ADN complémentaire à l'autre. Les perfectionnements portent notamment sur la réduction du nombre de cycles d'amplification, le recours à une opération de digestion des nucléases avant l'amplification, un nouvel adaptateur oligonucléotide permettant la mise en oeuvre du procédé perfectionné, et enfin de nouveaux procédés d'amplification sélective des fragments uniques voulus avec dégradation sélective des fragments contenant des séquences communes aux deux populations. Les fragments d'une population échantillon sont amplifiés en utilisant une amorce qui confère aux produits d'amplification une résistance à la dégradation des nucléases. Puis les fragments d'une population témoin sont amplifiés en utilisant une amorce qui cible les produits d'amplification en vue d'une dégradation préférentielle. On a recours à plusieurs cycles d'hybridation, de traitement des nucléases et d'amplification jusqu'à obtenir un enrichissement des fragments spécifique de la population échantillon.
Lindemann Garrett W.
Schueler Paula
Boehringer Mannheim Corporation
Norton Rose Or S.e.n.c.r.l. S.r.l./llp
Roche Diagnostics Operations Inc.
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