Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions
View Patent ↗A method of fabricating a DNA molecule of user-defined sequence. The method comprises the steps of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an even or odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths. In one embodiment starting sequence fragments are of different lengths, n, n+1, n+2, etc.
1. A method of producing a DNA molecule of 1-10 kilobases of user-defined sequence from short oligos of length n (n-mers) comprising the steps of:
virtually preselecting a multiplicity of DNA sequence segments that will comprise said DNA molecule of user-defined sequence by using computational techniques to virtually break said user-defined sequence into virtual fragments of length n (n-mers) of defined size,
providing fragments in vitro by providing fragments of length n (n-mers) of defined size that correspond to said virtual fragments wherein said multiplicity of DNA sequence segments comprise oligos in multiple reading frames,
arraying fragments in vitro by arraying said fragments of length n (n-mers) of defined size into groups,
separating DNA sequence segments temporally in vitro by separating said DNA sequence segments of length n (n-mers) of defined size temporally, and
assembling groups in vitro by assembling said groups into double-strand DNA molecules of predetermined base-pairs using parallel synthesis, DNA shuffling, and DNA polymerase wherein said step of separating said DNA sequence segments temporally and said step of assembling said groups into double-strand DNA molecules of predetermined base-pairs is accomplished by said DNA sequence segments being added gradually, in an order that is predicted computationally to minimize errors, to produce said DNA molecule of user-defined sequence, and
wherein said step of assembling said groups into double-strand DNA molecules utilizes starting oligos of length n (n-mers) where n is an odd number.