METHODS AND APPARATUS FOR NUCLEIC ACID SYNTHESIS USING OLIGO-TEMPLATED POLYMERIZATION

• US 20150196917A1
• Filed: 01/26/2015
• Published: 07/16/2015
• Est. Priority Date: 08/02/2012
• Status: Abandoned Application

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Kay, Mark A., Barzel, Adi
• Application Number:
  US14/605,538
• Publication Number:
  US 20150196917A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/18 Transport of container or devices
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B01L 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2537/155 Cyclic reactions
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

A method and apparatus for synthesizing nucleic acid (NA) by sequential addition of nucleotide triphosphates to a single stranded NA molecule (growing-chain) is described. The growing-chain is annealed to an oligonucleotide, wherein the 3′ end of the oligonucleotide is complementary to the 3′ end of the growing-chain and wherein the protruding 5′ end of the oligonucleotide has the correct sequence to serve as a template for the growing-chain to produce a NA product. The growing-chain is then brought into contact with a polymerase in the presence of one or more nucleotide triphosphates under conditions permitting the polymerase to catalyze addition of one or more nucleotide triphosphates to the 3′ end of the growing-chain using the annealed oligonucleotide as a template. After polymerization has occurred, the annealed growing-chain and oligonucleotide are denatured so that the steps can be repeated until the NA product of desired length is synthesized.

Claims

1. An apparatus for synthesizing nucleic acid (NA), the apparatus comprising:

(a) an array of oligonucleotides, wherein said array comprises a plurality of oligonucleotides of a fixed length, and wherein the oligonucleotides collectively comprise every possible sequence permutation;

(b) a reaction chamber, wherein an oligonucleotide is contacted with a nucleic acid molecule growing-chain, said growing-chain having a 3′

end, said oligonucleotide having a 3′

end and a protruding 5′

end;

(c) wherein the 3′

end of the oligonucleotide is complementary to the 3′

end of the growing-chain;

(d) wherein the protruding 5′

end of the oligonucleotide has the correct sequence to serve as a template for the growing-chain to produce a NA product; and

(e) wherein the oligonucleotide and growing-chain are in the presence of one or more nucleotides under conditions permitting the polymerase to catalyze addition of one or more nucleotide triphosphates to the 3′

end of the growing-chain using the annealed oligonucleotide as a template;

(f) a denaturing chamber; and

(g) an automated arm configured for moving between the array of oligonucleotides, the elongation chamber, the denaturing chamber, and the reservoir of neutralization solution during a cycle of nucleic acid polymerization.

2. The apparatus of claim 1:

wherein the growing-chain is bound to a magnetic bead; and

wherein the magnetic bead is immobilized on the bottom of the elongation chamber.

3. The apparatus of claim 2, wherein the growing chain is bound to the magnetic bead by an attachment chemistry selected from the group consisting of streptavidin-biotin, alkyne-azide, and amine-NHS.

4. The apparatus of claim 1:

wherein the growing-chain is bound to magnetic bead;

wherein the magnetic bead is immobilized on a magnetized needle; and

wherein the needle is attached to a distal end of the automated arm.

5. The apparatus of claim 4, wherein the growing chain is bound to the magnetic bead by an attachment chemistry selected from the group consisting of streptavidin-biotin, alkyne-azide, and amine-NHS.

6. The apparatus of claim 1, wherein the oligonucleotides are bound to said reaction chamber.

7. The apparatus of claim 1, wherein one or more of the chambers is thermoregulated.

8. The apparatus of claim 1:

wherein the elongation chamber is subdivided into separate chambers; and

wherein each contains a different nucleotide triphosphate.

9. The apparatus of claim 1, further comprising a PCR chamber.

10. The apparatus of claim 1, wherein the fixed length of the oligonucleotides in the array ranges from 6 to 21 nucleotides.

11. The apparatus of claim 1, wherein the fixed length is 11 nucleotides.

12. The apparatus of claim 1, wherein the fixed length is 7 nucleotides.

13. An apparatus for synthesizing nucleic acid (NA), the apparatus comprising:

(a) an array of oligonucleotides, wherein said array comprises a plurality of oligonucleotides of a fixed length, and wherein the oligonucleotides collectively comprise every possible sequence permutation;

(b) a reaction chamber, wherein an oligonucleotide is contacted with a nucleic acid molecule growing-chain, said growing-chain having a 3′

end, said oligonucleotide having a 3′

end and a protruding 5′

end;

(c) wherein the 3′

end of the oligonucleotide is complementary to the 3′

end of the growing-chain;

(d) wherein the protruding 5′

end of the oligonucleotide has the correct sequence to serve as a template for the growing-chain to produce a NA product; and

(e) wherein the oligonucleotide and growing-chain are in the presence of one or more nucleotides under conditions permitting the polymerase to catalyze addition of one or more nucleotide triphosphates to the 3′

end of the growing-chain using the annealed oligonucleotide as a template;

(f) a denaturing chamber;

(g) an automated arm configured for moving between the array of oligonucleotides, the elongation chamber, the denaturing chamber, and the reservoir of neutralization solution during a cycle of nucleic acid polymerization; and

(h) a washing chamber.

14. The apparatus of claim 13:

wherein the growing-chain is bound to a magnetic bead; and

wherein the magnetic bead is immobilized on the bottom of the elongation chamber.

15. The apparatus of claim 14, wherein the growing chain is bound to the magnetic bead by an attachment chemistry selected from the group consisting of streptavidin-biotin, alkyne-azide, and amine-NHS.

16. The apparatus of claim 13:

wherein the growing-chain is bound to magnetic bead;

wherein the magnetic bead is immobilized on a magnetized needle; and

wherein the needle is attached to a distal end of the automated arm.

17. The apparatus of claim 16, wherein the growing chain is bound to the magnetic bead by an attachment chemistry selected from the group consisting of streptavidin-biotin, alkyne-azide, and amine-NHS.

18. The apparatus of claim 13, wherein the oligonucleotides are bound to said reaction chamber.

19. The apparatus of claim 13, wherein one or more of the chambers is thermoregulated.

20. The apparatus of claim 13:

wherein the elongation chamber is subdivided into separate chambers; and

wherein each contains a different nucleotide triphosphate.

21. The apparatus of claim 13, further comprising a PCR chamber.

22. The apparatus of claim 13, wherein the fixed length of the oligonucleotides in the array ranges from 6 to 21 nucleotides.

23. The apparatus of claim 13, wherein the fixed length is 11 nucleotides.

24. The apparatus of claim 13, wherein the fixed length is 7 nucleotides.