Method for carrying out the parallel sequencing of a nucleic acid mixture on a surface
First Claim
1. A method for sequencing in parallel at least two different nucleic acids present in a nucleic acid mixture, whereby a surface is produced comprising islands of nucleic acids of in each case the same type by the following steps:
- (a1) provision of a surface, on which at least primer molecules of a first primer and of a second primer, and, where appropriate, a nucleic acid mixture comprising the nucleic acid molecules with which both primers can hybridize, have been irreversibly immobilized, with the two primers forming a primer pair;
(a2) hybridisation of the nucleic acid molecules in the nucleic acid mixture with one or with both primers of the same primer pair;
(a3) extension of the irreversibly immobilized primer molecules in a complementary manner to the counterstrand, with the formation of secondary nucleic acids;
(a4) provision of the surface in a form which is freed from nucleic acid molecules which are not bound to the surface by irreversible immobilization;
(a5) amplification of the secondary nucleic acids in the formation of tertiary nucleic acids, whereby said islands of nucleic acids are defined as discrete locations of tertiary nucleic acids of in each case the same type and whereby the tertiary nucleic acids which are immobilzed on this surface are sequenced in parallel by the following steps;
(b) provision of counterstrands of the tertiary nucleic acids, i.e. TNCs, (c) extension of the TNCs by one nucleotide, with the nucleotide at the 2′
-OH position or at the 3′
-OH position carrying a protecting group which prevents further extension, the nucleotide carrying a molecular group which enables the nucleotide to be identified;
(d) identification of the incorporated nucleotide, (e) removal of the protecting group and removal or alteration of the molecular group of the incorporated nucleotide, which is used for identification, (f) repetition of step (c), and of the subsequent steps until the desired sequence information has been obtained.
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Abstract
The invention relates to a method for sequencing in parallel at least two different nucleic acids present in a nucleic acid mixture, characterized in that
(a) a surface is provided, which surface possesses islands of nucleic acids of in each case the same type, i.e. tertiary nucleic acids;
(b) counterstrands of the tertiary nucleic acids, i.e. TNCs, are provided;
(c) the TNCs are extended by one nucleotide, with
the nucleotide at the 2′-OH position or at the 3′-OH position carrying a protecting group which prevents further extension,
the nucleotide carrying a molecular group which enables the nucleotide to be identified;
(d) the incorporated nucleotide is identified;
(e) the protecting group is removed and the molecular group of the incorporated nucleotide, which is used for identification, is removed or altered, and
(f) step (c) and subsequent steps are repeated until the desired sequence information has been obtained.
100 Citations
19 Claims
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1. A method for sequencing in parallel at least two different nucleic acids present in a nucleic acid mixture, whereby a surface is produced comprising islands of nucleic acids of in each case the same type by the following steps:
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(a1) provision of a surface, on which at least primer molecules of a first primer and of a second primer, and, where appropriate, a nucleic acid mixture comprising the nucleic acid molecules with which both primers can hybridize, have been irreversibly immobilized, with the two primers forming a primer pair;
(a2) hybridisation of the nucleic acid molecules in the nucleic acid mixture with one or with both primers of the same primer pair;
(a3) extension of the irreversibly immobilized primer molecules in a complementary manner to the counterstrand, with the formation of secondary nucleic acids;
(a4) provision of the surface in a form which is freed from nucleic acid molecules which are not bound to the surface by irreversible immobilization;
(a5) amplification of the secondary nucleic acids in the formation of tertiary nucleic acids, whereby said islands of nucleic acids are defined as discrete locations of tertiary nucleic acids of in each case the same type and whereby the tertiary nucleic acids which are immobilzed on this surface are sequenced in parallel by the following steps;
(b) provision of counterstrands of the tertiary nucleic acids, i.e. TNCs, (c) extension of the TNCs by one nucleotide, with the nucleotide at the 2′
-OH position or at the 3′
-OH position carrying a protecting group which prevents further extension,the nucleotide carrying a molecular group which enables the nucleotide to be identified;
(d) identification of the incorporated nucleotide, (e) removal of the protecting group and removal or alteration of the molecular group of the incorporated nucleotide, which is used for identification, (f) repetition of step (c), and of the subsequent steps until the desired sequence information has been obtained. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification
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- Resources
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Current AssigneeAxaron Bioscience AG
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Original AssigneeAxaron Bioscience AG
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InventorsFischer, Achim
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Application NumberUS10/168,557Publication NumberTime in Patent OfficeDaysField of SearchUS Class Current435/6CPC Class CodesC12Q 1/6874 involving nucleic acid arra...C12Q 2521/313 Type II endonucleases, i.e....C12Q 2525/191 incorporating an adaptorC12Q 2525/301 Hairpin oligonucleotidesC12Q 2565/543 characterised by the use of...
