Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1995-05-19
1998-06-23
Jones, W. Gary
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 243, 435 6, 435 5, 435 911, 435 71, 435 772, 435 79, 435 912, 216 43, 216 48, 216 51, 216 41, 216 55, 216 65, 216 66, 216 75, 264139, 264176, 422 57, 422 61, C07H 2100, C07H 2104, C12Q 168, G01N 2100
Patent
active
057707219
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The invention relates to molecular biology, and particularly to a method to prepare matrices for detecting mismatches.
BACKGROUND OF THE INVENTION
Known in the art is a method for preparing matrices containing a set of oligonucleotides of specified length: A, T, G, C, C, T, . . . etc. (PCT/GB 89/00460, 1989). Such matrices are useful in the detection of mismatches.
In the above method, oligonucleotides are synthesized directly on a glass support by successively masking certain regions of the glass surface with the strips of varied width made of silicon rubber.
For example, masking can be effected in the following way.
A first set of four bases, A, T, G, and C, is obtained by laying four wide strips on a rectangular glass plate (support).
A second set is produced by superimposing four identical strips in an orthogonal fashion, thereby getting a matrix consisting of 16 dinucleotides. Thereafter a third and a forth layer is put down, each consisting of four narrow strips (a fourth part of the width of the first strips).
In so doing, each layer of the narrow strips is put within one layer of wider strips. The final matrix contains a 256-membered set of tetranucleotides.
This process is repeated again and again, each time putting two perpendicular layers of narrow strips onto underlying two layers times of strips which are four time as large. Each layer adds one base to the oligonucleotide sequence and increases four-fold the number of unique sequences in the set.
The final size of the resulting matrix is determined by the width of said strips. For example, when strips as narrow as 1 mm wide are used, the matrix consisting of 256 oligonucleotides will occupy a space of about .about.256 mm.sup.2. With increasing number of oligonucleotides, the matrix becomes correspondingly large and will eventually become inconvenient to use. In particular it may be difficult to monitor simultaneously all cells, especially when fluorescent labeling is used.
The above method consists of many steps and is therefore time-consuming.
Another method for preparing matrices containing a set of oligonucleotides for detecting mismatches comprises providing a matrix containing a set of oligonucleotides of known length comprising the steps of coating a solid support gel layer; removing parts of said gel layer to conform it to the prescribed topology of the matrix so that the resulting gel spots are spaced from one another and their number is equal to the number of oligonucleotides in the set; and finally immobilizing presynthesized oligonucleotides from the set ATTGCC . . . to each gel spot, one by one (PCT/RU 92/00052, 1992). In that method, partial removal of gel to form "spacers"on the matrix is effected by mechanical scribing using a special stencil.
The above technology is less time-consumms since microdoses of presynthesized oligonucleotides are immobilized in the gel simultaneously, in one operation.
Moreover, for a given output signal the three-dimensional gel matrix will be smaller than the one that has been formed by synthesizing oligonucleotides directly in the near-surface layer of support.
However, mechanical scribing of the gel does not allow to obtain cells less than about 100 .mu.m-per side, due to the mechanical damage caused to the gel edges (more or less rounded angles, possible peeling of gel from the solid support, etc.) which leads to deformation of the matrix topology and decreases the meaningful signal, down to total absence of the latter.
DISCLOSURE OF THE INVENTION
The present invention aims at developing a method to manufacture matrices for detection of mismatches, wherein shaping of the gel layer should be performed in accordance with the matrix topology in such a way as to ensure obtaining, at a high precision, the gel squares of a side from 10 to 50 .mu.m safely attached to the support which, in turn, will allow one to miniaturize the matrix and simultaneously increase the number of its elements.
To realize the above goals, the known method to manufacture matrices dedicated to the
REFERENCES:
patent: 4050898 (1977-09-01), Goffe et al.
patent: 4912032 (1990-03-01), Hoffman et al.
patent: 5212050 (1993-05-01), Mier et al.
Ershov Gennady Moiseevich
Mirzabekov Andrei Darievich
Jones W. Gary
Rees Dianne
University of Chicago
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