Nucleic acid affinity columns
First Claim
1. A nucleic acid amplification template array comprising a surface to which are attached at least 50 nucleic acid probes having different predetermined nucleic acid sequences and wherein:
- i) each different probe is localized in a known location of said surface;
ii) the density of said probes is greater than about 60 different probes per 1 cm2;
and iii) said different probes have an identical terminal 3′
nucleic acid sequence and an identical terminal 5′
nucleic acid sequence, said identical terminal 3′ and
5′
nucleic acid sequence capable of serving as primer binding sites.
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Accused Products
Abstract
This invention provides nucleic acid affinity matrices that bear a large number of different nucleic acid affinity ligands allowing the simultaneous selection and removal of a large number of preselected nucleic acids from the sample. Methods of producing such affinity matrices are also provided. In general the methods involve the steps of a) providing a nucleic acid amplification template array comprising a surface to which are attached at least 50 oligonucleotides having different nucleic acid sequences, and wherein each different oligonucleotide is localized in a predetermined region of said surface, the density of said oligonucleotides is greater than about 60 different oligonucleotides per 1 cm2, and all of said different oligonucleotides have an identical terminal 3′ nucleic acid sequence and an identical terminal 5′ nucleic acid sequences; b) amplifying said multiplicity of oligonucleotides to provide a pool of amplified nucleic acids; and c) attaching the pool of nucleic acids to a solid support.
83 Citations
11 Claims
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1. A nucleic acid amplification template array comprising a surface to which are attached at least 50 nucleic acid probes having different predetermined nucleic acid sequences and wherein:
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i) each different probe is localized in a known location of said surface;
ii) the density of said probes is greater than about 60 different probes per 1 cm2;
andiii) said different probes have an identical terminal 3′
nucleic acid sequence and an identical terminal 5′
nucleic acid sequence, said identical terminal 3′ and
5′
nucleic acid sequence capable of serving as primer binding sites.- View Dependent Claims (2, 3, 4, 5, 6)
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7. A nucleic acid affinity matrix that reduces known nucleic acid molecules in a sample, said affinity matrix comprising a multiplicity of at least 20 different predetermined nucleic acid probes, wherein:
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there exists in said affinity matrix a probe complementary to each of said nucleic acid molecules or a subsequence thereof;
said at least 20 different predetermined probes do not include every possible probe having the same length as said predetermined probes; and
wherein the probes comprising said multiplicity of probes are preselected by the steps of;
i) determining an allowable Tm interval;
ii) determining a mismatch Tm threshold;
iii) identifying all nucleic acid sequences of a preselected length complementary to a known nucleic acid molecule whose Tm to said known nucleic acid molecule is within said allowable Tm interval;
iv) determining the likelihood of each of said nucleic acid sequences complementary to said known nucleic acid molecule also occurring in an unknown message;
v) sorting said sequences in order of likelihood with the least likely sequence first;
vi) selecting the first nucleic acid sequence whose Tm to all other known nucleic acid molecules in said sample is below said mismatch Tm;
vii) repeating step vi) until a desired number of nucleic acids that specifically hybridize, under stringent conditions, to said known nucleic acid molecule are obtained;
viii) repeating steps iii) through vii) until at least one nucleic acid sequence that hybridizes specifically under stringent conditions to each known nucleic acid molecule is selected. - View Dependent Claims (8, 9, 10)
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11. A nucleic acid affinity matrix that reduces known nucleic acid molecules in a sample, said affinity matrix comprising a multiplicity of at least 20 different predetermined nucleic acid probes, wherein:
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there exists in said affinity matrix a probe complementary to each of said nucleic acid molecules or a subsequence thereof; and
said at least 20 different probes do not include every possible probe having the same length as said predetermined probes; and
wherein said affinity matrix is prepared according to a method that comprises providing a nucleic acid amplification template array comprising a surface to which are attached at least 20 template nucleic acid probes having different predetermined nucleic acid sequences, wherein;
i) each different template probe is localized in a known location of said surface;
ii) the density of said template probes is greater than about 60 different template probes per 1 cm2; and
iii) said different template probes have an identical terminal 3′
nucleic acid sequence and an identical terminal 5′
nucleic acid sequence; and
amplifying said multiplicity of oligonucleotides at least about 10 fold to provide a nucleic acid pool from which the at least 20 different predetermined nucleic acid probes are obtained.
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Specification