Method and Device For the Synthesis and the Analysis of Suppert-Bound Arrays of Oligomers, Especially of Primer Pairs for PCR, as well as Oligomer-Carrying Supports
First Claim
1. A method for synthesis of a support-bound array (1) of oligomers, in particular oligonucleotides (2) or oligoribonucleotides, with free ends A, in particular with free 3′
- OH ends (3), whereby a temporary protecting group (4) is provided on end B, preferably on the 5′
OH end (5) of the oligomer, and permanent protecting groups (6) are provided on reactive side groups, characterised, in that the temporary protecting group (4) is removed from the end B, in particular from the 5′
OH end (5), on completion of the combinatorial synthesis of the oligomers (2) on the support (9), in that the free ends B, in particular the 5′
OH ends (5), are then cross-linked via cross-linking (7) even before the permanent protecting groups (6) are removed, in that the covalent bond (8) of the synthesised oligomers (2), in particular of the oligonucleotides (2) or oligoribonucleotides (2) can then be split off via the end A, in particular the 3′
OH end, onto the support (9) by a portion, preferably by a predominant portion of the synthesised oligomer (2), resulting in free ends A, in particular free 3′
OH ends (3), in that also a portion, preferably a predominant portion of the synthesised oligomers (2), binds covalently (8) to the support (9), due to the abovementioned cross-linking (7) at the end B, in particular at the 5′
OH end (5), after removal of the ends A, in particular the 3′
OH ends (3), from the support (9).
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Accused Products
Abstract
For synthesis of a support-bound array of oligomers with free ends A, whereby a temporary protecting group is provided on the 5′-OH end of the oligomers and permanent protecting groups on reactive side groups, it is proposed that the temporary protecting group is removed from the end B after combinatorial synthesis of the oligomers onto the support, the free ends B are then cross-linked even before splitting of the permanent protecting groups, then the covalent bond of the synthesised oligomers can be removed via the end A onto the support, resulting in free ends A, and a portion of the synthesised oligomers binds covalently to the support also after removal of the ends A from the support.
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Citations
21 Claims
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1. A method for synthesis of a support-bound array (1) of oligomers, in particular oligonucleotides (2) or oligoribonucleotides, with free ends A, in particular with free 3′
- OH ends (3), whereby a temporary protecting group (4) is provided on end B, preferably on the 5′
OH end (5) of the oligomer, and permanent protecting groups (6) are provided on reactive side groups,characterised, in that the temporary protecting group (4) is removed from the end B, in particular from the 5′
OH end (5), on completion of the combinatorial synthesis of the oligomers (2) on the support (9),in that the free ends B, in particular the 5′
OH ends (5), are then cross-linked via cross-linking (7) even before the permanent protecting groups (6) are removed,in that the covalent bond (8) of the synthesised oligomers (2), in particular of the oligonucleotides (2) or oligoribonucleotides (2) can then be split off via the end A, in particular the 3′
OH end, onto the support (9) by a portion, preferably by a predominant portion of the synthesised oligomer (2), resulting in free ends A, in particular free 3′
OH ends (3),in that also a portion, preferably a predominant portion of the synthesised oligomers (2), binds covalently (8) to the support (9), due to the abovementioned cross-linking (7) at the end B, in particular at the 5′
OH end (5), after removal of the ends A, in particular the 3′
OH ends (3), from the support (9).- View Dependent Claims (3, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20)
characterised, in that an array to be examined is placed into close contact with the heating block (11), in that the array (1) is covered with a translucent planar film or plate (13) to avoid evaporation of a reaction buffer, in that the array (1) for parallel analysis of PCR reactions is irradiated with excitation light (14), in particular with UV light, in that the data recorded by the detection unit (17) are transferred to a commercial computer, where they undergo image analysis. -
14. The method for analysis of the arrays (1) of oligomers synthesised as claimed in claim 1 to 10, using a device as claimed in claim 12, characterised in that the arrays (1) are analysed using the device mentioned in claim 12 above.
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15. A support having at least one array of oligomers with free 3′
- OH ends for parallel analysis of PCR reactions, characterised in that the array was applied to the support as claimed in any one of the methods described in claims 1 to 10.
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16. The support as claimed in claim 15 with an oligonucleotide library, characterised in that the oligonucleotide library has been created by combinatorial synthesis of a limited number of suitable monomers.
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17. The support as claimed in claim 15 or 16 with an oligonucleotide library, characterised in that the oligonucleotide library is present as a 2-dimensional array on the support derived appropriately, whereby the individual components are assigned to the oligonucleotide library as defined by location.
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18. The support as claimed in any one of claims 15 to 17 with an oligonucleotide library, characterised in that the oligonucleotide library has a free 3′
- OH end, which is a substrate of template-dependent DNA polymerases or RNA polymerases after hybridisation of template DNA.
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19. The support as claimed in any one of claims 15 to 18 with an oligonucleotide library, characterised in that two defined oligonucleotides per defined location were synthesised in the oligonucleotide library.
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20. The support with at least one array of oligomers, which have undergone an in situ cleaning step, characterised in that the array was applied to the support according to a method described as claimed in claims 1 to 10.
- OH ends (3), whereby a temporary protecting group (4) is provided on end B, preferably on the 5′
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2. The method for synthesis of a support-bound array (1) of oligomers, in particular oligonucleotides (2) or oligoribonucleotides, with free A ends, in particular with free 3′
- OH ends (3), whereby a first temporary protecting group (4) is provided at the end B, in particular at the 5′
OH end (5) of the oligomers and permanent protecting groups (6) are provided on reactive side groups,characterised, in that in a first step following combinatorial synthesis of a first array (1) of oligomers (2) bound at specific, precisely defined locations (10) on the support (9) the first temporary protecting group (4) is removed at the end B, in particular at the 5′
-OH end (5) of the oligomers, and the now free ends B are then either cross-linked via cross-linking (7) or are blocked by a second permanent protecting group (30),in that in a second step a second temporary protecting group (20) different to the abovementioned first temporary protecting group 4) is removed on the support (9), resulting in a reactive group (22) on the support (9), in that in a third step a second array of precisely defined oligomers (2) is constructed on the reactive group (22) preferably by means of combinatorial synthesis, whose free ends B, in particular whose free 5′
-OH ends (5) can be cross-linked, as in the first step,in that in a fourth step the permanent protecting groups (6, 30) are split off, whereby the choice of suitable handles (8) determines that the majority of oligomers are present with a free end A, in particular with free 3′
-OH end (3).- View Dependent Claims (4)
- OH ends (3), whereby a first temporary protecting group (4) is provided at the end B, in particular at the 5′
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5. A method for synthesis of a support-bound array (1) of oligomers, in particular oligonucleotides or oligoribonucleotides, with free 3′
- OH ends (3), whereby a temporary protecting group (4) is provided at the 5′
OH end (5) of the oligomers and permanent protecting groups (6) are provided on reactive side groups,characterised, in that the temporary protecting group (4) is removed from the 5′
OH end (5) on completion of combinatorial synthesis,in that the free 5′
OH ends (5) are then attached via cross-linking (7) even before the permanent protecting groups (6) are removed,in that the covalent bond (8) is removed via the 3′
OH end of the synthesised oligomers onto the support (9) by a portion, preferably by a predominant portion of the synthesised oligomers, resulting in free 3′
OH ends (3), so that after the 3′
OH ends are removed from the support a portion, preferably a predominant portion of the synthesised oligomers, can bind covalently onto the support (9) due to cross-linking (7) at the 5′
OH end (5).
- OH ends (3), whereby a temporary protecting group (4) is provided at the 5′
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12. A device for analysis of arrays (1) of oligomers,
characterised, in that a heating block (11) is provided with a substantially planar contact surface, in that means are provided for irradiating excitation light (14), especially UV light, onto an array positioned on the heating block, in that a detection unit (17) is provided, in particular a CCD array for fluorescent light (19) emitted from the array of oligomers, in that provided between the detection unit (17) and the array (1) to be examined is a fluorescent light filter (18) which collimates the excitation light (14), but admits the emitted fluorescent light (19), and in that the abovementioned analysis is repeated, in particular as a time flow curve during an ongoing enzymatic reaction, in particular during a PCR reaction, which alters the abovementioned array of oligomers.
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21. The method for synthesis of a support-bound array (1) of oligomer pairs (21), especially of oligonucleotides (2) or oligoribonucleotides,
characterised, in that at least two oligomers (2) different to one another are synthesised or applied per defined location on an array (10), that both oligomers (2) have free 3′ - -OH ends (3) or are derived at free 3′
-OH ends (3), and that a high-grade parallelised polymerase chain reaction (PCR) is carried out with the array of oligomer pairs (21) and that during the PCR reaction a preferably thermostable enzymatic activity, in particular a helicase, a gyrase or topoisomerase, is added along with ATP which unravels superhelical twists.
- -OH ends (3) or are derived at free 3′
Specification