Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Synthesis of peptides
Reexamination Certificate
1998-11-19
2001-02-06
Marschel, Ardin H. (Department: 1631)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Synthesis of peptides
C530S334000, C536S025300, C536S025310, C422S050000, C422S068100
Reexamination Certificate
active
06184347
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the preparation of high-density arrays of surface-bound oligonucleotides by a step-wise synthesis of oligonucleotides on the surface of a substrate and, in particular, to a method and system for precisely confining the application of reactive deoxynucleoside phosphoramidites to particular cells within the high-density array.
BACKGROUND OF THE INVENTION
A combination of synthetic chemical technologies and certain computer-related technologies has lead to the development of an important analytical tool in the field of molecular biology commonly referred to as the “gene chip.” Gene chips are high-density arrays of oligonucleotides bound to a chemically prepared substrate such as silicon, glass, or plastic. Each cell, or element, within the array is prepared to contain a single oligonucleotide species, and the oligonucleotide species in a given cell may differ from the oligonucleotide species in the remaining cells of the high-density array. Gene chips may be used in DNA hybridization experiments in which radioactively, fluorescently, or chemiluminescently labeled DNA or RNA molecules are applied to the surface of the gene chip and are bound, via Watson-Crick base pair interactions, to specific oligonucleotides bound to the gene chip. The gene chip can then be analyzed by radiometric or optical methods to determine to which specific cells of the gene chip the labeled DNA or RNA molecules are bound. Thus, in a single experiment, a DNA or RNA molecule can be screened for binding to tens or hundreds of thousands of different oligonucleotides.
Hybridization experiments can be used to identify particular gene transcripts in mRNA preparations, to identify the presence of genes or regulatory sequences in cDNA preparations, or to sequence DNA and RNA molecules. Particularly in the latter application, the effectiveness of employing gene chips depends of the precision with which specific oligonucleotides can be synthesized within discrete cells of the gene chip. As with any chemical synthetic process, various factors may cause the yields of specific steps in the synthesis of oligonucleotides to be less than 100%, leading to unintended and unwanted intermediate species. During an oligonucleotide lengthening step in the synthesis of oligonucleotides on the surface of a gene chip, reactive deoxynucleoside phosphoramidites are successively applied, in concentrations exceeding the concentrations of target hydroxyl groups of the substrate or growing oligonucleotide polymers, to specific cells of the high-density array. Then, unreacted deoxynucleoside phosphoramidites from multiple cells of the high-density array are washed away in a single wash step to prepare for a subsequent step of oligonucleotide synthesis. Unfortunately, during the wash step, unreacted deoxynucleoside phosphoramidites may migrate to regions outside the specific region of the high-density array to which they were applied and react with functional groups of the high-density array substrate or bound oligonucleotides rather than being cleanly removed from the surface of the gene chip. These unintended deoxynucleoside phosphoramidite reactions may result in the spreading, or blooming, of the deoxynucleoside phosphoramidite reaction to adjoining regions of the gene chip and may even lead to cross contamination of adjoining cells. As a consequence, the cells of the high-density array may end up containing a mixture of different oligonucleotides rather than a single specific oligonucleotide. A related blooming problem may occur when various phosphoramidite dyes are applied to the surface of the gene chip to mark specific cells or features. Blooming of these phosphoramidite dyes may lead to imprecise and low-resolution marking of cells and features.
Although a method for successively removing unreacted phosphoramidite reactants from individual cells of the high-density array might be envisioned, such successive treatment of individual cells would greatly increase the time required for preparation of gene chips, and would increase the complexity of the mechanical devices that are used to prepare gene chips. Instead, a need has been recognized in the area of gene chip manufacture for a method for bulk removal of unreacted phosphoramidite reactants from the cells of a high-density array without producing the blooming phenomenon resulting from reactions of phosphoramidite reactants outside the specific areas of a gene chip to which they are originally applied.
SUMMARY OF THE INVENTION
The present invention provides a method for the bulk removal of unreacted phosphoramidite reactants from the surface of a high-density array so that the unreacted phosphoramidite reactants do not react with reactive functional groups of the substrate of the high-density array or with chemical species bound to the substrate of the high-density array in areas outside of the specific area to which the phosphoramidite reactants are originally applied. In the method of the present invention, a phosphoramidite-reactive solution is employed to wash unreacted phosphoramidites from the surface of the high-density array. The phosphoramidite-reactive solution reacts with, and deactivates, unreacted phosphoramidites before they have a chance to react with reactive functional groups of either the substrate of the high-density array or of chemical species bound to the substrate of the high-density array. Use of the present invention thus allows for the preparation of high-density arrays with densities, or resolutions, determined by the precision with which the phosphoramidite reactants can be applied to the surface of the high-density array.
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Kanemoto Roy H.
Perbost Michel G. M.
Agilent Technologie,s Inc.
Marschel Ardin H.
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