Compositions and Methods For High Fidelity Assembly of Nucleic Acids

• US 20130059296A1
• Filed: 08/23/2012
• Published: 03/07/2013
• Est. Priority Date: 08/26/2011
• Status: Abandoned Application

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Jacobson, Joseph, Schindler, Daniel, Lawton, Scott S.
• Application Number:
  US13/592,827
• Publication Number:
  US 20130059296A1
• Time in Patent Office: Days
• US Class Current: 435/6.1
• 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: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/1089 Design, preparation, screening or analysis of libraries using computer algorithms
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/20 Sequence assembly

Abstract

Aspects of the invention relate to methods, compositions and algorithms for designing and producing a target nucleic acid. The method can include: (1) providing a plurality of blunt-end double-stranded nucleic acid fragments having a restriction enzyme recognition sequence at both ends thereof; (2) producing via enzymatic digestion a plurality of cohesive-end double-stranded nucleic acid fragments each having two different and non-complementary overhangs; (3) ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase; and (4) forming a linear arrangement of the plurality of cohesive-end double-stranded nucleic acid fragments, wherein the unique arrangement comprises the target nucleic acid. In certain embodiments, the plurality of blunt-end double-stranded nucleic acid fragments can be provided by: releasing a plurality of oligonucleotides synthesized on a solid support; and synthesizing complementary strands of the plurality of oligonucleotides using a polymerase based reaction.

Claims

31. A method for designing a plurality of starting nucleic acids to be assembled into a target nucleic acid, the method comprising:

(a) obtaining an input target sequence of a target nucleic acid;

(b) selecting a plurality of subsequences therein such that every two adjacent subsequences overlap with each other by N bases;

(c) storing the resulting overlapping N-base sequences in a memory;

(d) comparing the overlapping N-base sequences to one another to ensure that they differ from one another by at least one base; and

(e) repeating steps (b) to (d) until a plurality of satisfactory nucleic acid fragments are obtained wherein any two adjacent starting nucleic acid fragments uniquely overlap with each other by N bases.

35. The method of claim 31, wherein the target nucleic acid is a non-naturally occurring nucleic acid.

36. The method of claim 31, wherein the target nucleic acid is at least 500, 800, 1000, 1500, 2000, or 3000 bases long.

37. The method of claim 31, wherein each subsequence is about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300 or more bases long.

38. The method of claim 31, wherein N is an integral and is 3, 4, 5, 6, 7, 8, or more.

39. A plurality of starting nucleic acids to be assembled into a target nucleic acid, designed according to the method of claim 31.

42. A system for assembling a target nucleic acid, the system comprising:

a solid support for synthesizing the plurality of starting nucleic acids of claim 31, wherein each starting nucleic acid further comprises an engineered universal primer binding site and an engineered restriction enzyme recognition sequence;

a polymerase reaction unit for synthesizing complementary strands of the plurality of starting nucleic acids a polymerase-based reaction using a universal primer complementary to the universal primer binding site, thereby producing a plurality of blunt-end double-stranded nucleic acid fragments;

a digestion unit for producing a plurality of cohesive-end double-stranded nucleic acid fragments via enzymatic digestion of the plurality of blunt-end double-stranded nucleic acid fragments, wherein the plurality of cohesive-end double-stranded nucleic acid fragments each have two different and non-complementary overhangs; and

a ligation unit for ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase, wherein a first overhang of a first cohesive-end double-stranded nucleic acid fragment is uniquely complementary to a second overhang of a second cohesive-end double-stranded nucleic acid fragment.

32-1. The method of claim 32, wherein the restriction enzyme recognition site is a type IIS recognition site.

40. The plurality of starting nucleic acids of claim 40, each further comprising an engineered universal primer binding site for amplifying the plurality of starting nucleic acids therefrom.

41. The plurality of starting nucleic acids of claim 40, each further comprising an engineered restriction enzyme recognition sequence.

43. A computer program product for designing a plurality of starting nucleic acids to be assembled into a target nucleic acid, said program residing on a hardware computer readable storage medium and having a plurality of instructions which, when executed by a processor, cause the processor to perform operations comprising:

(a) obtaining a target sequence of a target nucleic acid;

(b) selecting a plurality of subsequences therein such that every two adjacent subsequences overlap with each other by N bases;

(c) storing the resulting overlapping N-base sequences in a memory;

(d) comparing the overlapping N-base sequences to one another to ensure that they differ from one another by at least one base; and

(e) repeating steps (b) to (d) until a plurality of satisfactory starting nucleic acids are obtained wherein any two adjacent starting nucleic acids uniquely overlap with each other by N bases.