Claims
- 1. A method of synthesizing a DNA molecule of user-defined sequence, comprising the steps of:
providing a pre-defined, double-stranded, sequence of DNA with a single-stranded overhang, and lengthening said pre-defined, double-stranded, sequences of DNA by the addition of user-selected, single stranded DNA sequences.
- 2. The method of synthesizing a DNA molecule of user-defined sequence of claim 1, including the step of tethered said pre-defined, double-stranded, sequence of DNA with a single-stranded overhang.
- 3. The method of synthesizing a DNA molecule of user-defined sequence of claim 1, including the step of tethered said pre-defined, double-stranded, sequence of DNA with a bead.
- 4. The method of synthesizing a DNA molecule of user-defined sequence of claim 1, including the step of ligating user-selected, single stranded DNA sequences to lengthen said surface-tethered, pre-defined, double-stranded, sequence of DNA.
- 5. The method of synthesizing a DNA molecule of user-defined sequence of claim 1, including the step of adding a final user-selected, single stranded DNA sequences creating a final overhang and ligating a pre-defined double-stranded sequence which has a single-stranded overhang complementary to said final overhang to said final user-selected, single stranded DNA sequence.
- 6. The method of synthesizing a DNA molecule of user-defined sequence of claim 1 wherein said user-selected, single stranded DNA sequence anneals to said pre-defined, double-stranded, sequence of DNA with a single-stranded overhang by way of hydrogen bonding to said single-stranded overhang.
- 7. The method of synthesizing a DNA molecule of user-defined sequence of claim 1 wherein said pre-defined, double-stranded, sequence of DNA with a single-stranded overhang is approximately 30 base pairs long.
- 8. The method of synthesizing a DNA molecule of user-defined sequence of claim 1 wherein said pre-defined, double-stranded, sequence of DNA with a single-stranded overhang is a T7 primer.
- 9. The method of synthesizing a DNA molecule of user-defined sequence of claim 1 wherein said pre-defined, double-stranded, sequence of DNA with a single-stranded overhang is a T3 primer.
- 10. The method of synthesizing a DNA molecule of user-defined sequence of claim 1 wherein said pre-defined, double-stranded, sequence of DNA with a single-stranded overhang is a SP6 primer.
- 11. The method of synthesizing a DNA molecule of user-defined sequence of claim 1 wherein said single-stranded overhang is a three bases overhang.
- 12. The method of synthesizing a DNA molecule of user-defined sequence of claim 1 wherein said user-selected, single stranded DNA sequences are oligos of six bases.
- 13. The method of synthesizing a DNA molecule of user-defined sequence of claim 1 wherein said pre-defined, double-stranded, sequence of DNA and said preselected, single stranded DNA sequences are determined by using computational techniques to break said DNA molecule of user-defined sequence into fragments of defined size.
- 14. The method of synthesizing a DNA molecule of user-defined sequence of claim 1, including the step of ligating user-selected, single stranded DNA sequences to lengthen said surface-tethered, pre-defined, double-stranded, sequence of DNA using temperatures from 0 degrees C to 70 degrees C.
- 15. The method of synthesizing a DNA molecule of user-defined sequence of claim 1, including the steps of ligating user-selected, single stranded DNA sequences to lengthen said surface-tethered, pre-defined, double-stranded, sequence of DNA and using ligases comprising T4 DNA ligase.
- 16. The method of synthesizing a DNA molecule of user-defined sequence of claim 1, including the steps of ligating user-selected, single stranded DNA sequences to lengthen said surface-tethered, pre-defined, double-stranded, sequence of DNA and using ligases comprising T7 RNA ligase.
- 17. The method of synthesizing a DNA molecule of user-defined sequence of claim 1, including the steps of ligating user-selected, single stranded DNA sequences to lengthen said surface-tethered, pre-defined, double-stranded, sequence of DNA and using ligases comprising Ampligase thermostable DNA ligase.
- 18. The method of synthesizing a DNA molecule of user-defined sequence of claim 1, including the steps of ligating user-selected, single stranded DNA sequences to lengthen said surface-tethered, pre-defined, double-stranded, sequence of DNA and using ligases comprising T4 DNA ligase and/or T7 RNA ligase and/or Ampligase thermostable DNA ligase.
- 19. The method of synthesizing a DNA molecule of user-defined sequence of claim 1, wherein said steps of lengthening said pre-defined, double-stranded, sequences of DNA by the addition of user-selected, single stranded DNA sequences comprises sequentially combining reactions in which a first pre-defined sequence of DNA is lengthened by the addition of additional pre-defined sequences of DNA and sequentially combining reactions in which said first pre-defined sequence of DNA is lengthened by the addition of additional pre-defined sequences of DNA.
- 20. The A method of synthesizing a DNA molecule of user-defined sequence of claim 19 wherein said sequentially combining reactions in which a first pre-defined sequence of DNA is lengthened by the addition of additional pre-defined sequences of DNA and sequentially combining reactions in which said first pre-defined sequence of DNA is lengthened by the addition of additional pre-defined sequences of DNA are performed in parallel.
- 21. A method of-synthesizing a DNA molecule of user-defined sequence, comprising the steps of:
providing a first pre-defined sequence of DNA, sequentially combining reactions in which said first pre-defined sequence of DNA are lengthened by the addition of additional pre-defined sequences of DNA, and sequentially combining reactions in which said additional pre-defined sequences of DNA are lengthened by the addition of yet additional pre-defined sequences of DNA.
- 22. The A method of synthesizing a DNA molecule of user-defined sequence of claim 21 wherein said steps of sequentially combining reactions in which said first pre-defined sequences of DNA are lengthened by the addition of additional pre-defined sequences of DNA and sequentially combining reactions in which said additional pre-defined sequences of DNA are lengthened by the addition of yet additional pre-defined sequences of DNA are performed in parallel.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 60/367,929 filed Mar. 25, 2002 titled “Voltage-Controlled Method for Constructing User-Defined, Very Long DNA Sequences” and U.S. Provisional Patent Application No. 60/367,928 filed Mar. 25, 2002 titled “Method for Constructing User-Defined, Very Long DNA Sequences.” U.S. Provisional Patent Application No. 60/367,929 filed Mar. 25, 2002 titled “Voltage-Controlled Method for Constructing User-Defined, Very Long DNA Sequences” and U.S. Provisional Patent Application No. 60/367,928 filed Mar. 25, 2002 titled “Method for Constructing User-Defined, Very Long DNA Sequences” are incorporated in this application by this reference.
CONSTRUCTING USER-DEFINED, LONG DNA SEQUENCES
[0002] The United States Government has rights in this invention pursuant to Contract No. W-7405-ENG-48 between the United States Department of Energy and the University of California for the operation of Lawrence Livermore National Laboratory.
Provisional Applications (2)
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Number |
Date |
Country |
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60367929 |
Mar 2002 |
US |
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60367928 |
Mar 2002 |
US |