Replica amplification of nucleic acid arrays
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.
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.
2. The method of claim 1 wherein said immobilized array of spots of a nucleic acid capture activity are arranged in a predetermined geometry.
3. The method of claim 1 wherein said spots of nucleic acid capture activity are aligned with other microfabricated features.
4. A method of making a plurality of nucleic acid arrays wherein said nucleic acid arrays are produced according to the method of claim 1 .
5. The method of claim 1 further including the step of amplifying one or more of the immobilized nucleic acid molecules to produce amplified nucleic acid molecules.
6. The method of claim 5 further including the step of immobilizing the amplified nucleic acid molecules.
7. The method of claim 5 wherein the step of amplifying is carried out by thermal-cycled polymerase chain reaction, isothermal self-sustained sequence replication or DNA ligase amplification reaction.
8. The method of claim 1 further including the step of determining the sequence of one or more of the immobilized nucleic acid molecules.
9. The method of claim 5 further comprising the step of transferring the amplified nucleic acid molecules to a second substrate by contacting the second substrate to the first substrate.
10. A method of making a nucleic acid molecule array comprising:
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.
11. The method of claim 10 wherein one or more of the nucleic acid capture activities are arranged in a predetermined geometry.
12. The method of claim 10 wherein one or more of the nucleic acid capture activities are aligned with other microfabricated features.
13. The method of claim 10 further including the step of amplifying one or more of the immobilized nucleic acid molecules to produce amplified nucleic acid molecules.
14. The method of claim 13 further including the step of immobilizing the amplified nucleic acid molecules.
15. The method of claim 13 wherein the step of amplifying is carried out by thermal-cycled polymerase chain reaction, isothermal self-sustained sequence replication or DNA ligase amplification reaction.
16. The method of claim 10 further including the step of determining the sequence of one or more of the nucleic acid molecules.
17. The method of claim 13 further comprising the step of transferring the amplified nucleic acid molecules to a second substrate by contacting the second substrate to the first substrate.