Synthetic ligation reassembly in directed evolution
First Claim
1. A method of producing a progeny library comprised of chimerized but pre-determined polynucleotide sequences each of which is comprised of a pre-determined number of building block sequences that are assembled in non-random order, the method comprising:
- (a) generating a plurality of pre-determined nucleic acid building block sequences comprised of sequences delineated by demarcation points selected from aligned progenitor nucleic acid sequences; and
(b) non-stochastically assembling said nucleic acid building block sequences to produce said chimerized but pre-determined polynucleotide sequences, such that a designed overall assembly order is achieved for each of said chimerized but pre-determined polynucleotide sequence.
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Abstract
Harvesting the full richness of biodiversity is instantly recognized by Diversa Corporation as a powerful means to access both novel molecules having direct commercial utility as well as molecular templates that could be retooled to acquire commercial utility. A directed evolution process for rapid and facilitated production from a progenitor polynucleotide template, of a library of mutagenized progeny polynucleotides wherein each of the 20 naturally encoded amino acids is encoded at each original codon position. This method, termed site-saturation mutagenesis, or simply saturation mutagenesis, is preferably based on the use of the degenerate N,N,G/T sequence. Also, a method of non-stochastically producing a library of chimeric nucleic acid molecules having an overall assembly order that is chosen by design. Accordingly, a set of progenitor templates, such as genes (e.g. a family of esterase genes) or genes pathways (e.g. encoding antibiotics) can be shuffled to generate a sizable library of distinct progeny polynucleotide molecules (e.g. 10100) and correspondingly encoded polypeptides. Screening of these polynucleotide libraries enables the identification of a desirable molecular species that has a desirable property, such as a specific enzymatic activity serviceable for a commercial application, or a novel antibiotic. Also, a method of retooling genes and gene pathways by the introduction of regulatory sequences, such as promoters, that are operable in an intended host, thus conferring operability to a novel gene pathway when it is introduced into an intended host. For example a novel man-made gene pathway, generated based on microbially-derived progenitor templates, that is operable in a plant cell.
38 Citations
25 Claims
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1. A method of producing a progeny library comprised of chimerized but pre-determined polynucleotide sequences each of which is comprised of a pre-determined number of building block sequences that are assembled in non-random order, the method comprising:
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(a) generating a plurality of pre-determined nucleic acid building block sequences comprised of sequences delineated by demarcation points selected from aligned progenitor nucleic acid sequences; and (b) non-stochastically assembling said nucleic acid building block sequences to produce said chimerized but pre-determined polynucleotide sequences, such that a designed overall assembly order is achieved for each of said chimerized but pre-determined polynucleotide sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method of producing a progeny library comprised of chimerized but pre-determined polynucleotide sequences each of which is comprised of a pre-determined number of building block sequences that are assembled in non-random order, the method comprising:
- (a) generating a plurality of pre-determined nucleic acid building block sequences comprised of sequences delineated by demarcation points selected to create nucleic acid building blocks of a pre-determined size from aligned progenitor nucleic acid sequences, wherein each of said plurality of pre-determined nucleic acid building blocks is a double stranded building block with two nucleotide overhangs generated by a method comprising the steps of (i) generating overlapping blunt-ended amplification products;
(ii) melting the blunt-ended amplification products to produce single stranded nucleic acids;
(iii) annealing the single stranded nucleic acids to produce a population of double stranded nucleic acids; and
(iv) selecting for double stranded nucleic acids with two nucleotide overhangs;
wherein selecting for double stranded nucleic acids with two nucleotide overhangs comprises degrading other nucleic acids in the population with a 3′
acting nuclease; and
(b) non-stochastically assembling said nucleic acid building block sequences to produce said chimerized but pre-determined polynucleotide sequences, such that a designed overall assembly order is achieved for each of said chimerized but pre-determined polynucleotide sequence. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
- (a) generating a plurality of pre-determined nucleic acid building block sequences comprised of sequences delineated by demarcation points selected to create nucleic acid building blocks of a pre-determined size from aligned progenitor nucleic acid sequences, wherein each of said plurality of pre-determined nucleic acid building blocks is a double stranded building block with two nucleotide overhangs generated by a method comprising the steps of (i) generating overlapping blunt-ended amplification products;
Specification