Method and system for polynucleotide synthesis

• US 7,164,992 B1
• Filed: 03/22/2002
• Issued: 01/16/2007
• Est. Priority Date: 03/22/2001
• Status: Active Grant

• Current Assignee: Blue Heron Biotech, LLC (Eurofins Scientific SE)
• Original Assignee: Blue Heron Biotechnology Incorporated
• Inventors: Mulligan, John T., Tabone, John C., Brickner, R. Gregg
• Primary Examiner(s): Brusca; John S.
• Application Number:
  US10/104,986
• Time in Patent Office: 1,761 Days
• Field of Search: 702 19- 20, 703/11, 707/102
• US Class Current: 702/20
• CPC Class Codes: G16B 99/00 Subject matter not provided for in other groups of this subclass
• CPC Class Codes: G16C 20/10 Analysis or design of chemical reactions, syntheses or processes

Abstract

Methods and systems for automated polynucleotide synthesis design are provided. Example embodiments provide an Automated Polynucleotide Synthesis Design System (“APSDS”), which automatically generates a synthesis design for a designated target sequence specification. In one embodiment, the APSDS comprises a synthesis design engine, user interface support, a synthesis rules data repository, and a synthesis data repository. The APSDS automatically generates a synthesis design by receiving a target sequence(s) specification, generating a potential synthesis design, evaluating the potential design against synthesis rules, and when the evaluation indicates that the potential design is not successful according to the synthesis rules, adjusting the design to generate a new potential synthesis design and repeating the process of evaluating and adjusting until a potential synthesis design is found that satisfies the synthesis rules or until no solution is found.

Claims

1. A method in a computer system for automated generation of a design for synthesizing a designated target double-stranded polynucleotide molecule according to a convergent synthesis technique, comprising:

decomposing the designated target molecule into a plurality of potential double-stranded fragments, each fragment having an associated fragment definition and having ends that will potentially ligate with ends of other fragments;

automatically determining whether the potential double-stranded fragments satisfy a plurality of synthesis criteria for synthesizing fragments and generating an indication of the determination;

when the indication indicates that the potential fragments do not satisfy the synthesis criteria, automatically adjusting the fragment definitions of the plurality of potential fragments thereby adjusting at least one joint of the designated target molecule and repeating the automatically determining whether the potential fragments satisfy the plurality of synthesis criteria, the generating the indication of determination, and the automatically adjusting the fragment definitions until the generated indication indicates that the plurality of potential fragments will synthesize properly to create the designated polynucleotide molecule; and

generating a design output that indicates the polynucleotide sequences of the plurality of potential fragments as defined by the adjusted fragment definitions and an ordering for combining the plurality of polynucleotide sequences according to a convergent synthesis technique.

2. The method of claim 1 further comprising using the generated design output to automatically control a polynucleotide synthesizer apparatus.

3. The method of claim 1 wherein a fragment definition comprises, for each strand of the associated sequence fragment, an indication of a position in the designated target molecule, an indication of a nucleotide sequence, and an indication of length.

4. The method of claim 1 wherein the automatically adjusting the fragment definitions comprises adjusting at least one of overhang type, overhang length, joint position between a plurality of fragments, or a sequence of nucleotides in at least one strand of the fragment.

5. The method of claim 4 wherein overhang type is adjusted prior to the sequence.

6. The method of claim 4 wherein overhang type is adjusted prior to joint position.

7. The method of claim 4 wherein the sequence is adjusted prior to overhang type.

8. The method of claim 4 wherein the sequence is adjusted prior to joint position.

9. The method of claim 1 wherein the automatically determining whether the potential fragments satisfy the plurality of synthesis criteria incorporates ends-checking rules to determine the ligatability of a plurality of groups of potential fragments.

10. The method of claim 1 wherein the synthesis criteria specify empirically-based ligation rules.

11. The method of claim 1 wherein the synthesis criteria comprises rules that check for approximate complementarity between base pairs of fragment overhangs of a group of potential fragments.

12. The method of claim 11 wherein the rules determine full matches between the base pairs of the fragment overhangs.

13. The method of claim 11 wherein the rules determine partial matches between the base pairs of the fragment overhangs.

14. The method of claim 13 wherein a partial match between two fragment overhangs occurs when three of four base pairs of the respective fragment overhangs are complementary and at least one of three bases of the fragment overhang of the first fragment is approximately complementary to one of three bases of the fragment overhang of the second fragment.

15. The method of claim 13 wherein a partial match between two fragment overhangs occurs when three of four base pairs of the respective fragment overhangs are complementary.

16. The method of claim 13 wherein a partial match between two fragment overhangs occurs when six of nine base pairs of the respective fragment overhangs are complementary.

17. The method of claim 11 wherein approximate complementarity between base pairs of two fragment overhangs is detected when at least one of an C-T base pair and an G-T base pair is present.

18. The method of claim 11 wherein approximate complementarity between base pairs of two fragment overhangs is detected when a first base is empirically found under synthesis conditions to behave as a recognized mate of a second base but is not a recognized mate, where A and T are recognized mates and G and C are recognized mates, and the first base is present in the first fragment overhang and the second base is present in the second fragment overhang.

19. The method of claim 1 wherein the synthesis criteria are dynamically determined.

20. The method of claim 1 wherein the synthesis criteria comprise ends-checking rules that are derived empirically.

21. The method of claim 1 wherein the synthesis criteria comprise a rule for determining the presence of G or C base in mating positions when the overhangs of two fragments are annealed together.

22. The method of claim 1 wherein the indication of the determination of whether the potential fragments satisfy the synthesis criteria generates a score.

23. The method of claim 22 wherein a separate score is generated each time the fragment definitions are adjusted.

24. A computer-readable memory medium containing instructions for controlling a computer processor, when executed, to automatically generate a design for synthesizing a designated target double-stranded polynucleotide molecule according to a convergent synthesis technique, by performing a method comprising:

decomposing the designated target molecule into a plurality of potential double-stranded fragments, each fragment having an associated fragment definition and having ends that will potentially ligate with ends of other fragments;

automatically determining whether the potential double-stranded fragments satisfy a plurality of synthesis criteria for synthesizing fragments and generating an indication of the determination;

when the indication indicates that the potential fragments do not satisfy the synthesis criteria, automatically adjusting the fragment definitions of the plurality of potential fragments thereby adjusting at least one joint of the designated target molecule and repeating the automatically determining whether the potential fragments satisfy the plurality of synthesis criteria, generating the indication of the determination and the automatically adjusting the fragment definitions until the generated indication indicates that the plurality of potential fragments will synthesize properly to create the designated polynucleotide molecule; and

generating a design output that indicates the polynucleotide sequences of the plurality of potential fragments as defined by the adjusted fragment definitions and an ordering for combining the plurality of polynucleotide sequences according to a convergent synthesis technique.

25. The memory medium of claim 24 further comprising using the generated design output to automatically control a polynucleotide synthesizer apparatus.

26. The memory medium of claim 24 wherein a fragment definition comprises, for each strand of the associated sequence fragment, an indication of a position in the designated target molecule, an indication of a nucleotide sequence, and an indication of length.

27. The memory medium of claim 24 wherein the automatically adjusting the fragment definitions comprises adjusting at least one of overhang type, overhang length, joint position between a plurality of fragments, or a sequence of nucleotides in at least one strand of the fragment.

28. The memory medium of claim 24 wherein the automatically determining whether the potential fragments satisfy the plurality of synthesis criteria incorporates rules to determine the ligatability of a plurality of groups of potential fragments.

29. The memory medium of claim 28 wherein the rules comprise at least one of empirically-based ligation rules, rules that check for approximately complementarity between base pairs of fragment overhangs of a group of potential fragments, rules that are dynamically determined, or ends-checking rules that are derived empirically.

30. The memory medium of claim 29 wherein the rules that check for approximate complementarity determine full matches between the base pairs of fragment overhangs.

31. The memory medium of claim 29 wherein the rules that check for approximate complementarity determine partial matches between the base pairs of fragment overhangs.

32. The memory medium of claim 24 wherein the indication of the determination of whether the potential fragments satisfy the synthesis criteria generates a score.

33. A computer system for generating a design for synthesizing a designated target double-stranded polynucleotide molecule according to a convergent synthesis technique, comprising:

a synthesis rule data repository that contains a plurality of synthesis criteria for synthesizing fragments according to the convergent synthesis technique;

a synthesis design engine that is configured to,

decompose the designated target molecule into a plurality of potential double-stranded fragments, each fragment having an associated fragment definition and having ends that will potentially ligate with ends of other fragments;

retrieve at least one of the plurality of synthesis criteria from the synthesis rule data repository;

automatically determine whether the potential double-stranded fragments satisfy the at least one synthesis criteria and generate an indication of the determination;

when the indication indicates that the potential fragments do not satisfy the synthesis criteria, automatically adjust the fragment definitions of the plurality of potential fragments thereby adjusting at least one joint of the designated target molecule and repeat the automatic determination of whether the potential fragments satisfy the synthesis criteria, the generation of the indication of the determination, and the automatic adjustment of the fragment definitions until the generated indication indicates that the plurality of potential fragments will synthesize properly to create the designated polynucleotide molecule; and

generate a design output that indicates the polynucleotide sequences of the plurality of potential fragments as defined by the adjusted fragment definitions and an ordering for combining the plurality of polynucleotide sequences according to a convergent synthesis technique; and

a synthesis design data repository that receives and stores the generated design output.

34. The computer system of claim 33 further comprising a control system that receives the generated design output from synthesis design data repository and uses generated design output to automatically control a polynucleotide synthesizer apparatus.

35. The computer system of claim 33 wherein a fragment definition comprises, for each strand of the associated sequence fragment, an indication of a position in the designated target molecule, an indication of a nucleotide sequence, and an indication of length.

36. The computer system of claim 33 wherein the automatic adjustment of the fragment definitions comprises adjusting at least one of overhang type, overhang length, joint position between a plurality of fragments, or a sequence of nucleotides in at least one strand of the fragment.

37. The computer system of claim 33 wherein the plurality of synthesis criteria for synthesizing fragments contained in the synthesis rule data repository incorporates rules that can be used to determine the ligatability of a plurality of groups of potential fragments.

38. The computer system of claim 37 wherein the rules comprise at least one of empirically-based ligation rules, rules that check for approximately complementarity between base pairs of fragment overhangs of a group of potential fragments, rules that are dynamically determined, or ends-checking rules that are derived empirically.

39. The computer system of claim 38 wherein the rules that check for approximate complementarity determine full matches between the base pairs of fragment overhangs.

40. The computer system of claim 38 wherein the rules that check for approximate complementarity determine partial matches between the base pairs of fragment overhangs.

41. The computer system of claim 33 wherein the generated indication of the determination of whether the potential fragments satisfy the synthesis criteria generates a score.