Method of generating translationally active linear dna...

C - Chemistry – Metallurgy – 12 – N

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C12N 15/10 (2006.01) C12N 15/11 (2006.01) C12Q 1/68 (2006.01) G01N 33/50 (2006.01)

Patent

CA 2596758

This invention provides a method for producing expressionally and translationally active linear DNA molecules that can be used as protein expression cassettes and are useful for high throughput protein expression and analysis in living cells. The method comprises a PCR amplification using two primers complementary to the sequences flanking the DNA sequence of interest, such as a cDNA, an open reading frame or a gene, that exists in expression vectors. The resultant PCR product contains a promoter, the DNA sequence of interest, and a termination sequence. The invention further provides different uses of these translationally active DNA cassettes, such as in proteome arrays.

La présente invention décrit un procédé qui permet de produire des molécules d~ADN linéaire, actives tant sur le plan de l~expression que de la traduction, qui peuvent être employées comme des cassettes d~expression de protéines et s~avèrent utiles lors de l~expression et l~analyse de protéines à haut rendement dans les cellules vivantes. Ce procédé comprend une amplification en chaîne par polymérase qui utilise deux amorces complémentaires aux séquences adjacentes à la séquence d~ADN recherchée, telle qu~un ADNc, un cadre de lecture ouvert ou un gène, qui existe dans les vecteurs d~expression. Le produit obtenu à la suite de l~amplification en chaîne par polymérase contient un promoteur, la séquence d~ADN recherchée et un terminateur. Cette invention permet également différentes utilisations de ces cassettes d~ADN actives sur le plan de la traduction, et ce dans les réseaux de protéomes par exemple.

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