Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions
A method 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 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.
1. A method of fabricating a DNA molecule from n-base oligonucleotides that are assembled into double-strand DNA molecules using DNA polymerase, comprising the steps of:
preselecting a multiplicity of DNA sequence segments that will comprise said DNA molecule, wherein said multiplicity of DNA sequence segments are of size n, where n is an odd number, wherein said multiplicity of DNA sequence segments comprises oligos in multiple reading frames,
separating said multiplicity of DNA sequence segments temporally, thereby providing a multiplicity of temporally separated DNA sequence segments, and
combining said multiplicity of temporally separated DNA sequence segments with at least one polymerase enzyme with overlaps between said temporally separated DNA sequence segments of size n, where n is an odd number, wherein said multiplicity of temporally separated DNA sequence segments join to produce said DNA molecule.