Chemistry: molecular biology and microbiology – Apparatus – Mutation or genetic engineering apparatus
Reexamination Certificate
2000-05-01
2001-08-14
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Apparatus
Mutation or genetic engineering apparatus
C435S006120, C435S091100, C435S091200, C435S283100, C435S285200, C435S287200, C536S023100, C536S024300, C536S024310, C536S025320, C386S349000
Reexamination Certificate
active
06274373
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of gene sequencing. More particularly, this invention relates to a gene sequencer, a high density bio-compact disk useful therewith and a method of sample preparation therefor. The high-density bio-compact disk and the sample preparation methodology find application in the field of oligonucleotide sequencing and DNA sequencing and detection generally.
SUMMARY OF THE INVENTION
In one aspect, the present invention features a sample preparation method for obtaining n-mer oligonucleotides from a sample containing oligonucleotide fragments comprising: (a) forming a solid support having all possible n-mer oligonucleotides attached to the surface of the support; (b) contacting the solid support resulting from step (a) with the sample under conditions causing the sample oligonucleotides to hybridize with the complementary n-mer oligonucleotides on the solid support; (c) contacting the solid support resulting from step (b) with a hydrolyzing agent; (d) separating the unbound oligonucleotides from the hybridized oligonucleotides; and (e) denaturing the hybridized n-mer oligonucleotides to obtain the n-mer oligonucleotides of the sample; wherein n is an integer selected from the integers 4-10,000, most advantageously 6-28.
In another aspect, the invention features a method of obtaining n-mer oligonucleotides from a sample containing oligonucleotide fragments comprising: (a) contacting a solid support adapted to couple with oligonucleotides in the sample with at least a portion of the sample; (b) contacting the solid support resulting from step (a) with a mixture of n-mer oligonucleotides for a time sufficient for the n-mer oligonucleotides to hybridize with the complementary n-mer oligonucleotides on the solid support; (c) separating the hybridized n-mer oligonucleotides from the unhybridized oligonucleotides; (d) denaturing the hybridized n-mer oligonucleotides to obtain the n-mer oligonucleotides complementary to those present in the sample; wherein n is an integer selected from the integers 4-10,000, most advantageously 6-28.
In still another aspect, the sample preparation method includes a method of obtaining n-mer oligonucleotides from a sample containing oligonucleotide fragments comprising: (a) contacting a solid support having bound thereon oligonucleotides from a sample with a mixture of a plurality of oligonucleotides having (k+m)-mers, wherein k+m=n, with a mixture of a plurality of first oligonucleotides, each being a k-mer and being without a free hydroxyl group at the 3′-end thereof, and a plurality of second oligonucleotides, each being a m-mer and being without a free phosphate group at the 5′-end thereof; (b) ligating the oligonucleotides on the solid support resulting from step (a); (c) removing the unligated oligonucleotides from the solid support; and (d) denaturing the hybridized n-mer oligonucleotides remaining on the solid support to obtain the n-mer oligonucleotides complementary with those present in the sample; wherein m, k and n are each an integer selected from the integers from 6-10,000, most advantageously 12-40, with the proviso that k+m=n.
In yet another aspect, the sample preparation method includes a method of obtaining n-mer oligonucleotides from a sample containing oligonucleotide fragments comprising: (a) contacting a solid support having bound thereon a plurality of oligonucleotides from a sample with a mixture of a plurality of h-mer oligonucleotides each having a phosphate group at both the 3′- and 5′-end, a plurality of i-mer oligonucleotides each having a hydroxyl, amino or thiol group at the 3′-end and no terminal phosphate group, and a plurality of j-mer oligonucleotides having a hydroxyl, amino or thiol group at the 5′-end and no terminal phosphate group; (b) chemically or enzymatically ligating the oligonucleotides on the solid support resulting from step (a); (c) removing the unligated oligonucleotides from the solid support resulting from step (b); and (d) denaturing the hybridized n-mer oligonucleotides remaining on the solid support to obtain the n-mer nucleotides complementary with those present in the sample; wherein h, i and j are each an integer selected from the integers from 6-10,000, most advantageously 18-60, with the proviso that h+i+j=n.
In yet another aspect of this invention, an assay element is described comprising a substrate having a surface including a plurality of discrete areas on the surface adapted to attach to a spacer molecule; a plurality of spacer molecules attached at a first end to said surface in each of the discrete areas, each of said spacer molecules adapted to being attached at its second end to a metallic surface or a label, each of said spacer molecules having a site between its first end and its second end capable of being cleaved; a first n-mer oligonucleotide having a first sequence attached to substantially all of the spacer molecules between the cleavage site and the first end of the spacer molecule, and a second n-mer oligonucleotide having a second sequence attached to substantially all of the spacer molecules; wherein substantially no other discrete areas on the surface of the substrate contain spacer molecules having n-mer oligonucleotides having the first sequence attached thereto and n is an integer selected from the integers 4-10,000, most advantageously 6-28.
The present invention also encompasses a method for determining the sequence of a (p+q+r)-mer segment of a gene suspected of being present in a sample comprising: (a) forming a solution of the sample and a mixture of q-mer oligonucleotides having all possible sequences of a q-mer oligonucleotide, or, optionally, a subset of all such possible sequences; (b) contacting an assay element with at least a portion of the solution of step (a), the assay element having a surface and plurality of spacer molecules bound to the surface, the spacer molecules having a first end bound to the surface and a second end bound to a metallic surface or a label and a cleavage site intermediate between the first and second ends, the spacer molecules further having a first p-mer oligonucleotide attached thereto between the cleavage site and the first end and a second r-mer oligonucleotide attached thereto between the cleavage site and the second end, the combination of p-mers and r-mers including all combinations of oligonucleotide sequences of p-mer and r-mer oligonucleotides, or, optionally, a subset of all such combinations, each particular combination of sequences of the p-mer and r-mer oligonucleotides being at a predetermined location on the surface; (c) ligating the resultant hybridized oligonucleotides attached to the spacer molecules resulting from step (b) above; (d) detecting the presence or absence of a particular sequence combination of the hybridized oligonucleotides at each predetermined location on the surface; and (e) processing the sequence information obtained from step (d) to deduce the sequence of the (p+q+r)-mer oligonucleotide present in the sample, wherein p, q and r are integers selected from the integers 4-10,000, most advantageously 6-26, and (p+q+r) does not exceed 30,000, and most advantageously 60. Steps (a)-(e) can be performed in parallel for different, multiple segments of a gene.
REFERENCES:
patent: 5412087 (1995-05-01), McGall et al.
patent: 5863708 (1999-01-01), Zanzucchi
patent: 0 721 062 A2 (1996-07-01), None
patent: WO 95/00530 (1995-01-01), None
patent: WO 95/09248 (1995-04-01), None
patent: WO 98/01533 (1998-01-01), None
Burstein Technologies, Inc.
Horlick Kenneth R.
Siew Jeffrey
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