COMPOSITIONS AND METHODS FOR HIGH FIDELITY ASSEMBLY OF NUCLEIC ACIDS
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.
1 . A method of producing a target nucleic acid having a predefined sequence, the method comprising:
providing a plurality of blunt-end double-stranded nucleic acid fragments having a restriction enzyme recognition sequence at both ends of each of the plurality of blunt-end double-stranded nucleic acid fragments;
producing a plurality of cohesive-end double-stranded nucleic acid fragments that together comprises the target nucleic acid sequence 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;
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; and
forming a linear arrangement of the plurality of cohesive-end double-stranded nucleic acid fragments, wherein the unique arrangement comprises the target nucleic acid having a predefined sequence.
2 . The method of claim 1 , wherein the plurality of blunt-end double-stranded nucleic acid fragments are generated from a plurality of single-stranded oligonucleotides immobilized on a solid support.
3 . The method of claim 1 , wherein the plurality of blunt-end double-stranded nucleic acid fragments comprises:
releasing a plurality of oligonucleotides synthesized on a solid support; and
synthesizing complementary strands of the plurality of oligonucleotides using a polymerase based reaction.
4 . The method of claim 3 , wherein the plurality of oligonucleotides each comprise a universal primer binding site, and wherein a universal primer complementary to the universal primer binding site is used in the polymerase based reaction.
5 . The method of claim 4 , wherein the plurality of oligonucleotides each comprise the restriction enzyme recognition sequence.
6 . The method of claim 5 , wherein the restriction enzyme recognition sequence is part of the universal primer binding site and is located at the 5′ or 3′ end of the universal primer binding site or the restriction enzyme recognition sequence is located upstream or downstream to the universal primer binding site.
7 . The method of claim 4 , wherein the universal primer has an affinity tag to facilitate affinity removal of undesirable enzymatic digestion products.
8 . (canceled)
9 . The method of claim 1 , wherein the plurality of blunt-end double-stranded nucleic acids comprises at least 3, 4, 5, 6, 7, 8, 10, 15 or 20 different blunt-end double-stranded nucleic acid fragments, and wherein each of the plurality of blunt-end double-stranded nucleic acid fragments is at least 50, 100, 200, or 300 bases long.
10 . (canceled)
11 . The method of claim 1 , wherein the restriction enzyme recognition sequence is the same for all blunt-end double-stranded nucleic acid fragments.
12 . The method of claim 1 , wherein the plurality of blunt-end double-stranded nucleic acid fragments comprise at least two different restriction enzyme recognition sequences recognizable by two different restriction enzymes that are selected to produce overhangs having the same number of bases.
13 . The method of claim 1 , wherein the restriction enzyme recognition sequence is capable of being recognized by a type IIs restriction enzyme.
14 . The method of claim 13 , wherein the type IIs restriction enzyme is BsaI, BsmBI, BspQI, BtgZI, BsmFI, FokI, BbvI, any variant thereof, or any combination thereof.
15 . The method of claim 1 , wherein the plurality of cohesive-end double-stranded nucleic acid fragments are designed such that the cohesive end in a cohesive-end double-stranded nucleic acid fragment is uniquely complementary to a next cohesive end in an adjacent cohesive-end double-stranded nucleic acid fragment.
16 . The method of claim 1 , wherein the overhangs are at least 3, 4, 5, 6, 7, or 8 bases long, and wherein the overhangs differ from one another by at least 1, 2, 3 or 4 bases.
17 .- 18 . (canceled)
19 . The method of claim 1 , further comprising, before the ligating step, purifying the plurality of cohesive-end double-stranded nucleic acid fragments to remove undesirable enzymatic digestion products.
20 .- 21 . (canceled)
22 . The method of claim 1 , wherein the ligase is T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, any variant thereof, or any combination thereof.
23 .- 24 . (canceled)
25 . The method of claim 1 , further comprising amplifying the target nucleic acid using a pair of primers specific to the target nucleic acid and a polymerase.
26 . (canceled)
27 . The method of claim 1 , wherein the plurality of blunt-end double-stranded nucleic acid fragments are hierarchically assembled from synthetic oligonucleotides.
28 . The method of claim 1 wherein the plurality of nucleic acid fragments are ligated in a single pool.
29 . The method of claim 1 wherein the plurality of nucleic acid fragments are in at least two pools, each nucleic acid fragment of the first pool having a terminal end complementary to a nucleic acid fragment of the second pool.
30 .- 44 . (canceled)