Methods And Compositions For Generating Mixtures Of Nucleic Acid Molecules

• US 20090075840A1
• Filed: 09/18/2007
• Published: 03/19/2009
• Est. Priority Date: 09/18/2007
• Status: Abandoned Application

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Leproust, Emily Marine, Myerson, Joel
• Application Number:
  US11/857,380
• Publication Number:
  US 20090075840A1
• Time in Patent Office: Days
• US Class Current: 506/17
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 50/18 using a particular method of attachment to the solid support
• CPC Class Codes: C40B 80/00 Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers

Abstract

In some embodiments, the present disclosure provides methods of making a mixture of nucleic acid molecules, the methods comprising the steps of: synthesizing on a substrate a population of nucleic acid molecules wherein each synthesized nucleic acid molecule comprises a substrate-attached proximal nucleic acid molecule, a distal nucleic acid molecule, and a cleavable linker linking the proximal nucleic acid molecule to the distal nucleic acid molecule, and harvesting distal nucleic acid molecules from the substrate by cleaving the cleavable linker under conditions that do not release the proximal nucleic acid molecule. Related compositions and kits are also provided.

Claims

1. A method for synthesizing nucleic acid molecules, said method comprising the steps of:

a) synthesizing an array of proximal nucleic acid molecules on a substrate,

b) incorporating a cleavable linker by contacting the array of proximal nucleic acid molecules with a cleavable phosphoramidite building block of formula I comprising;

2. The method of claim 1, wherein one of said W and Z comprises S and the other of said W and Z comprises O, and wherein said O and said Lc′

comprise an ester.

3. The method of claim 1 wherein the proximal nucleic acid molecules are bound to the substrate surface by an attachment linkage, and wherein the attachment linkage is devoid of a cleavable moiety.

4. The method of claim 1, wherein the proximal nucleic acid molecules are 2 to 30 nucleotide residues in length.

5. The method of claim 1, wherein the distal nucleic acid molecules are 10 to 500 nucleotide residues in length.

6. The method of claim 1, wherein the proximal nucleic acid molecules comprise the same base.

7. The method of claim 1, wherein said substrate comprises a non-porous glass surface.

8. The method of claim 1, wherein the method comprises using a pulse jet to deposit reagents at each of a plurality of sites in the array.

9. The method of claim 1, wherein step (d) comprises contacting the surface with a cleavage agent effective to cleave the cleavable linker Lc′

, said contacting being for a time and under conditions sufficient to result in cleaving the cleavable linker.

10. The method of claim 1, comprising recovering a solution phase mixture comprising the distal nucleic acids.

11. A method according to claim 1, wherein the distal nucleic acids released in step (d) each comprise a hydroxy at the 3′

position.

12. A composition comprising:

a modified substrate medium according to the following formula;

sm-PN₁-Lc′

-PN₂

wherein sm is a substrate medium;

wherein Lc′

comprises a cleavable linker obtained by incorporation of a cleavable phosphoramidite building block;

wherein PN₁ is a polynucleotide from 2-100 residues in length;

wherein PN₂ is a polynucleotide from 5 to 1000 residues in length;

wherein PN₁ is attached to the substrate medium by a non-cleavable attachment;

wherein said cleavable phosphoramidite building block comprises a compound of formula I;

13. The composition of claim 12, wherein all of the residues of PN₁ are the same.

14. The composition of claim 12, wherein said substrate medium comprises a planar medium.

15. The composition of claim 12, wherein said substrate comprises a bead.

16. The composition of claim 12, wherein said substrate medium comprises a planar medium and a plurality of features arranged in an array, wherein each feature comprises a plurality of identical PN₂ molecules.

17. A cleavable phosphoramidite building block of formula I comprising:

18. The method of claim 17, wherein one of said W and Z comprises S and the other of said W and Z comprises O, and wherein said O and said Lc′

together comprise an ester.

19. A kit for preparing a mixture of nucleic acids, comprising:

a) a cleavable phosphoramidite building block comprises a compound of formula I;

20. A kit for preparing a mixture of nucleic acids, comprising:

a) a modified substrate medium according to the following formula;

sm-PN₁-Lc-PN₂

wherein sm is a substrate medium;

wherein Lc′

comprises a cleavable linker obtained by incorporation of a cleavable phosphoramidite building block;

wherein PN₁ is a polynucleotide from 2-100 residues in length;

wherein PN₂ is a polynucleotide from 5 to 1000 residues in length;

wherein PN₁ is attached to the substrate medium by a non-cleavable linkage;

wherein said cleavable phosphoramidite building block comprises a compound of formula I;