Replica amplification of nucleic acid arrays
First Claim
1. A method of making an immobilized nucleic acid molecule array comprising:
- a) providing an immobilized array of spots of a nucleic acid capture activity selected from the group consisting of;
a hydrophobic compound;
a hydrophilic compound;
a compound which reacts covalently with a nucleic acid molecule;
a capture ligand;
an oligonucleotide;
an antibody;
a fragment of an antibody;
a protein;
a peptide;
an intercalator;
biotin;
avidin; and
streptavidin on a first substrate wherein;
i) said spots are separated by a distance greater than the diameter of said spots; and
ii) the size of said spots is less than the diameter of the excluded volume of said nucleic acid molecule to be captured; and
b) contacting said array of spots of a nucleic acid capture activity with an excess of nucleic acid molecules capable of being bound by said nucleic acid capture activity, said nucleic acid molecules having an excluded volume diameter greater than the diameter of said spots, resulting in an array of immobilized nucleic acid molecules in which said nucleic acid capture activity binds only one of said nucleic acid molecules having an excluded volume greater than the size of said spots.
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Accused Products
Abstract
Disclosed are improved methods of making and using immobilized arrays of nucleic acids, particularly methods for producing replicas of such arrays. Included are methods for producing high density arrays of nucleic acids and replicas of such arrays, as well as methods for preserving the resolution of arrays through rounds of replication. Also included are methods which take advantage of the availability of replicas of arrays for increased sensitivity in detection of sequences on arrays. Improved methods of sequencing nucleic acids immobilized on arrays utilizing single copies of arrays and methods taking further advantage of the availability of replicas of arrays are disclosed. The improvements lead to higher fidelity and longer read lengths of sequences immobilized on arrays. Methods are also disclosed which improve the efficiency of multiplex PCR using arrays of immobilized nucleic acids.
292 Citations
17 Claims
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1. A method of making an immobilized nucleic acid molecule array comprising:
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a) providing an immobilized array of spots of a nucleic acid capture activity selected from the group consisting of;
a hydrophobic compound;
a hydrophilic compound;
a compound which reacts covalently with a nucleic acid molecule;
a capture ligand;
an oligonucleotide;
an antibody;
a fragment of an antibody;
a protein;
a peptide;
an intercalator;
biotin;
avidin; and
streptavidin on a first substrate wherein;i) said spots are separated by a distance greater than the diameter of said spots; and ii) the size of said spots is less than the diameter of the excluded volume of said nucleic acid molecule to be captured; and b) contacting said array of spots of a nucleic acid capture activity with an excess of nucleic acid molecules capable of being bound by said nucleic acid capture activity, said nucleic acid molecules having an excluded volume diameter greater than the diameter of said spots, resulting in an array of immobilized nucleic acid molecules in which said nucleic acid capture activity binds only one of said nucleic acid molecules having an excluded volume greater than the size of said spots. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method of making a nucleic acid molecule array comprising:
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creating an array of nucleic acid capture activities selected from the group consisting of;
a hydrophobic compound;
a hydrophilic compound;
a compound which reacts covalently with a nucleic acid molecule;
a capture ligand;
an oligonucleotide;
an antibody or fragment of an antibody;
a protein;
a peptide;
an intercalator;
biotin; and
avidin or streptavidin on a first substrate,contacting the array of nucleic acid capture activities with an excess of nucleic acid molecules, binding the nucleic acid molecules to the nucleic acid capture activities wherein one or more nucleic acid capture activities bind only one nucleic acid molecule having an excluded volume preventing the binding of further nucleic acid molecules. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17)
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Specification