Methods for assembling nucleic acids

Inventors

Gill, John E. • Fu, Lixia • Gibson, Daniel G.

Assignees

Telesis Bio Inc

Abstract

The invention provides methods of assembling a DNA molecule having a desired sequence. The methods involve contacting a DNA ligase with a plurality of short oligonucleotides to be assembled and performing the ligase chain reaction to thereby generate a set of polynucleotides. Oligonucleotides in the plurality overlap with and are complementary to a sequence of at least one other oligonucleotide in the plurality, and at least 50% of the oligonucleotides in the plurality are 6-30 nucleotides in length. The set of polynucleotides produced are contacted with a DNA polymerase and dNTPs in a mixture to join the set of polynucleotides and thereby create a DNA molecule having a desired sequence by polymerase chain assembly. The method allows for production of oligonucleotides of any length having very high sequence fidelity to a desired sequence.

Core Innovation

The invention describes nucle-acid and DNA assembly that contacts a DNA ligase with short oligonucleotides comprising at least a portion of a desired sequence to create a mixture, wherein at least a portion of the short oligonucleotides overlap and are complementary to a portion of at least one other short oligonucleotide. When bound to their complementary sequence(s), at least two short oligos abut one another, enabling the ligase to process the abutting oligos. The resulting mixture is used to perform a ligase chain reaction (LCR) to thereby generate a set of polynucleotides.

The generated set of polynucleotides is then contacted with a DNA polymerase and dNTPs to join the set of polynucleotides and thereby assemble a DNA molecule having the desired sequence. The method is performed as a one-step reaction. In the disclosed implementations, the assembly is scarless and is performed in vitro/solution without requiring restriction enzymes or linker/adaptor/spacer DNA.

The approach is presented as generating high sequence fidelity without error-correction enzymes, with reported error rates and error-free construct percentages across multiple example target sizes and sequences. The disclosed assembly range includes constructs such as a 240 bp target, a 901 bp GFP subset, influenza HA (~1.9 kb) and NA (~1.6 kb), a ~10 kb product, and 200–1800 bp constructs spanning GC ranges. The disclosure emphasizes use of short overlapping oligonucleotides in the 8–22 nt range with best fidelity reported for 16–18 mers.

Claims Coverage

The independent claim covers a one-step ligase-chain-reaction-to-DNA-polymerase joining workflow that assembles a desired-sequence DNA molecule using short overlapping, complementary oligonucleotides whose bound forms abut one another. This claim requires contacting a DNA ligase with 8–25 nt oligonucleotides, performing an LCR to generate polynucleotides, and contacting the polynucleotides with a DNA polymerase and dNTPs to join and assemble the desired DNA, all as a one-step reaction. The dependent claims further refine quantitative constraints, restrict required components, and recite an error-rate threshold.

Across the independent claim and the highlighted dependent refinements, the inventive coverage centers on a one-step workflow in which overlapping, complementary short oligonucleotides that abut upon binding are used to generate polynucleotides by a ligase chain reaction, followed by polymerase and dNTP contacting to join polynucleotides into an assembled desired-sequence DNA molecule. Refinements shown in the partial claim set additionally constrain the assembled DNA length, the oligonucleotide length and quantity, exclude restriction enzymes, and impose an error-rate threshold.

Stated Advantages

Documented Applications