Method for making populations of defined nucleic acid molecules
View Patent ↗The present invention provides methods of making a population of nucleic acid molecules, wherein each nucleic acid molecule comprises a predetermined nucleic acid sequence, each of said methods comprising the steps of: (a) synthesizing, on a substrate, a population of nucleic acid molecules wherein: i) each synthesized nucleic acid molecule comprises a predetermined nucleic acid sequence; and ii) each synthesized nucleic acid molecule is localized to a defined area of said substrate; (b) harvesting said population of synthesized nucleic acid molecules from said substrate to yield harvested nucleic acid molecules; and (c) introducing said harvested nucleic acid molecules into vector molecules.
1. A method for making a population of nucleic acid molecules, the method comprising:
(a) harvesting a population of synthesized single-stranded nucleic acid molecules that are covalently attached to a microarray, wherein each of the synthesized nucleic acid molecules consists of a predetermined nucleic acid sequence consisting of:
(i) a 5′ primer binding region,
(ii) a 3′ primer binding region,
(iii) one of a number of different target sequences located between the 5′ and 3′ primer binding regions, and additionally,
(iv) one of a number of unique target identifier sequences,
wherein the target identifier sequence is not comprised within the target sequence or primer binding regions, and
wherein each unique target identifier sequence is associated with only one particular target sequence;
(b) amplifying the population of synthesized single-stranded nucleic acid molecules harvested in step (a) to generate a population of double-stranded DNA molecules, and
(c) introducing the double-stranded DNA molecules into vector molecules.
2. The method of claim 1 , wherein the synthesized nucleic acid molecules are covalently attached to the microarray via a linker.
3. The method of claim 1 , wherein the synthesized nucleic acid molecules have an average length of from about 20 nucleotides to about 100 nucleotides.
4. The method of claim 1 , wherein the microarray comprises a positionably addressable microarray.
5. The method of claim 1 , wherein the microarray comprises from about 1,000 to about 30,000 defined areas.
6. The method of claim 1 , wherein the harvesting comprises use of a hydrolyzing agent.
7. The method of claim 6 , wherein the hydrolyzing agent comprises ammonium hydroxide.
8. The method of claim 1 , wherein harvesting comprises cleavage of a photocleavable linker.
9. The method of claim 1 , wherein the target sequences comprise a nucleic acid library.
10. The method of claim 1 , wherein the unique target identifier sequence has a length of from about 4 nucleotides to about 8 nucleotides.
11. The method of claim 10 , wherein the unique target identifier sequence is 4 nucleotides long and has a sequence that is selected from the group consisting of the 256 nucleotide sequences that can be constructed from a 4 nucleotide sequence.
12. The method of claim 10 , wherein the unique target identifier sequence is 8 nucleotides long and has a sequence that is selected from the group consisting of the 65,536 nucleotide sequences that can be constructed from an 8 nucleotide sequence.
13. The method of claim 1 , wherein amplifying comprises a method selected from the group consisting of: polymerase chain reaction (PCR), ligase chain reaction, transcription amplification, self-sustained sequence replication, and nucleic acid based sequence amplification (NASBA).
14. The method of claim 1 , wherein both the 5′ primer binding region and the 3′ primer binding region have a length of from about 4 nucleotides to about 30 nucleotides.
15. The method of claim 1 , wherein the sequence of the 5′ primer binding region is the same in each of said synthesized nucleic acid molecules.
16. The method of claim 1 , wherein the sequence of the 3′ primer binding region is the same in each of said synthesized nucleic acid molecules.
17. The method of claim 1 , wherein the sequence of both the 3′ primer binding region and the 5′ primer binding region is the same in each of said synthesized nucleic acid molecules.
18. The method of claim 1 , wherein the 5′ primer binding region is located within the unique target identifier sequence.
19. The method of claim 1 , wherein the 3′ primer binding region is located within the unique target identifier sequence.
20. The method of claim 1 , wherein only synthesized nucleic acid molecules having one or more specific target identifier sequences are amplified.
21. The method of claim 1 , wherein the 3′ primer binding region comprises a restriction endonuclease recognition site.
22. The method of claim 1 , wherein the 5′ primer binding region comprises a restriction endonuclease recognition site.
23. The method of claim 1 , wherein each of said synthesized nucleic acid molecules further comprises an RNA polymerase promoter.
24. The method of claim 1 , wherein the vector molecules are expression vector molecules.
25. The method of claim 1 , wherein the vector molecules are selected from the group consisting of plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, and viral vectors.
26. A method for making a population of nucleic acid molecules, the method comprising:
(a) harvesting a population of synthesized single-stranded nucleic acid molecules that are covalently attached to a microarray, wherein each of the synthesized nucleic acid molecules consists of a predetermined nucleic acid sequence consisting of:
(i) a 5′ primer binding sequence,
(ii) a 3′ primer binding sequence,
(iii) one of a number of different target sequences located between the 5′ and 3′ primer binding regions, and additionally,
(iv) one of a number of unique target identifier sequences,
wherein the target identifier sequence is not comprised within the target sequence or primer binding regions, and
wherein each unique target identifier sequence is associated with a particular predetermined sub-population of target sequences;
(b) amplifying the population of synthesized single-stranded nucleic acid molecules harvested in step (a) to generate a population of double-stranded DNA molecules, and
(c) introducing the double-stranded DNA molecules into vector molecules.