Constructing user-defined, long DNA sequences

• US 20030186301A1
• Filed: 03/19/2003
• Published: 10/02/2003
• Est. Priority Date: 03/25/2002
• Status: Abandoned Application

• Current Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Tucker, James D., Christian, Allen T., Mariella, Raymond P. Jr.
• Application Number:
  US10/393,614
• Publication Number:
  US 20030186301A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation

Abstract

A system for synthesizing a DNA molecule of user-defined sequence. A pre-defined, double-stranded, sequence of DNA with a single-stranded overhang is provided. The pre-defined, double-stranded, sequence of DNA is lengthened by the addition user-selected, single stranded DNA sequences.

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.