Process for solid phase-sequencing of nucleic acid fragments

Claim 2

A process for sequencing a nucleic acid fragment by chemical degradation in a solid phase which comprises the steps of: (a) immobilizing labelled nucleic acid fragments in an aqueous solution on a solid support, said solid support having a high mechanical stability and anion exchange properties, as well as the ability to undergo a step of simultaneously cleaving said nucleic acid fragments immobilizing thereon and chemically eluting degraded nucleic acids from the solid support; (b) washing the labelled nucleic acid fragments immobilized on said solid support during step (a); (c) chemically modifying organic, heterocyclic nitrogen bases selected from the group consisting of thymine, quanine, cytosine, adenine and uracil, which form part of the nucleic acid fragments, so that a base specific chemical reaction corresponding to each nucleic acid residue in the sequence takes place; (d) washing the labelled nucleic acid fragments immobilized on the solid support and chemically modified during step (c); (e) sorting the labelled nucleic acid fragments; (f) simultaneously chemically cleaving the backbone of the nucleic acid fragments and chemically eluting degradated nucleic acids from the solid support; (g) lyophilizing the degradated nucleic acids; and (h) determining the sequence of the nucleic acid fragments by subjecting the degradated nucleic acids to electrophoresis.

Claim 3

A process for sequencing a nucleic acid fragment by chemical degradation in a solid phase which comprises the steps of: (a) immobilizing labelled nucleic acid fragments in an aqueous solution on a solid support, said solid support having a high mechanical stability and anion exchange properties, as well as the ability to undergo a step of simultaneously cleaving said nucleic acid fragments immobilized thereon and chemically eluting degraded nucleic acids from the solid support, said support comprising the reaction product of a macromolecular carrier which comprises: (i) 5 to 80% by volume of a macromolecular compound; (ii) 1 to 80% by volume of a macromolecular compound having 4,6-dihalogen-1,3,5-triazine groups; (iii) 0.5 to 50% by volume of a 2,4,6-trihalogen-1,3,5-triazine; (iv) up to 20% by volume of a halide; and (v) up to 5% by volume of buffering agents; and an amine salt of the Formula (I) ##STR3## wherein X is an NH 2 , OH or SH group; R is C 1 to C 10 alkyl, benzyl or a photochemically cleavable group selected from the group consisting of an o-nitrobenzene ester, a methylphenacyl ester and a heterocyclic group; R 1 , R 2 and R 3 are each hydrogen or C 1 to C 3 alkyl; and Y is halide or hydroxyl; (b) washing the labelled nucleic acid fragments immobilized on said solid support during step (a); (c) chemically modifying organic, heterocyclic nitrogen bases selected from the group consisting of thymine, guanine, cytosine, adenine and uracil, which form part of the nucleic acid fragments so that a base specific chemical reaction corresponding to each nucleic acid residue in the sequence takes place; (d) washing the labelled nucleic acid fragments immobilized on the solid support and chemically modified during step (c); (e) sorting the labelled nucleic acid fragments; (f) simultaneously chemically cleaving the backbone of the nucleic acid fragments and chemically eluting degradated nucleic acids from the solid support; (g) lyophilizing the degradated nucleic acids; and (h) determining the sequence of the nucleic acid fragments by subjecting the degradated nucleic acids to electrophoresis.

Claim 4

A process for sequencing a nucleic acid fragment by chemical degradation in a solid phase which comprises the steps of: (a) immobilizing labelled nucleic acid fragments in an aqueous solution on a solid support, said solid support having a high mechanical stability and anion exchange properties, as well as the ability to undergo a step of simultaneously cleaving said nucleic acid fragments immobilized thereon and chemically eluting degraded nucleic acids from the solid support, said support comprising the reaction product of a macromolecular carrier which comprises: (i) 5 to 80% by volume of a macromolecular compound; (ii) 1 to 80% by volume of a macromolecular compound having 4,6-dihalogen-1,3,5-triazine groups; (iii) 0.5 to 50% by volume of a 2,4,6-trihalogen-1,3,5-triazine; (iv) up to 20% by volume of a halide; and (v) up to 5% by volume of buffering agents; and an amine salt of the Formula (I) ##STR3## wherein X is an NH 2 , OH or SH group; R is C 1 to C 10 alkyl, benzyl or a photochemically cleavable group selected from the group consisting of an o-nitrobenzene ester, a methylphenacyl ester and a heterocyclic group; R 1 , R 2 and R 3 are each hydrogen or C 1 to C 3 alkyl; and Y is halide or hydroxyl; (b) washing the labelled nucleic acid fragments immobilized on said solid support during step (a); (c) chemically modifying organic, heterocyclic nitrogen bases selected from the group consisting of thymine, guanine, cytosine, adenine and uracil, which form part of the nucleic acid fragments so that a base specific chemical reaction corresponding to each nucleic acid residue in the sequence takes place; (d) washing the labelled nucleic acid fragments immobilized on the solid support and chemically modified during step (c); (e) sorting the labelled nucleic acid fragments; (f) simultaneously chemically cleaving the backbone of the nucleic acid fragments and chemically eluting degradated nucleic acids from the solid support; (g) lyophilizing the degradated nucleic acids; and (h) determining the sequence of the nucleic acid fragments by subjecting the degradated nucleic acids to electrophoresis.

Claim 5

The process for sequencing a nucleic acid fragment defined in claim 1 wherein the amine salt of the Formula (I) is prepared in situ by reacting a salt of the Formula (II) Y.sup.- --[NH.sub.3 --R--Y].sup.+ with a compound of the Formula (III) ##STR4##

Claim 6

The process for sequencing a nucleic acid fragment defined in claim 1 wherein in the salt of the Formula (I) X is amino; R is C 1 to C 10 alkyl or benzyl; and R 1 , R 2 and R 3 are each C 1 to C 3 alkyl.

Claim 7

The process for sequencing a nucleic acid fragment defined in claim 1 wherein the step of simultaneously chemically cleaving the nucleic acid backbone and chemically eluting the degradated nucleic acid fragments is carried out with an aqueous solution of piperidine.

Claim 8

The process for sequencing a nucleic acid fragment defined in claim 2 wherein in the salt of the Formula (I) X is amino, R is an o-nitrobenzene ester, R 1 , R 2 , and R 3 are each ethyl, and Y is bromide.