Computer-directed assembly of a polynucleotide encoding a target polypeptide
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Publication Number
US-7966338-B2
Publication Date
2011-06-21
Expiration Date
Abstract
The present invention outlines a novel approach to utilizing the results of genomic sequence information by computer-directed polynucleotide assembly based upon information available in databases such as the human genome database. Specifically, the present invention may be used to select, synthesize and assemble a novel, synthetic target polynucleotide sequence encoding a target polypeptide. The target polynucleotide may encode a target polypeptide that exhibits enhanced or altered biological activity as compared to a model polypeptide encoded by a natural (wild-type) or model polynucleotide sequence.
Core Innovation
The invention relates to a computer-directed workflow for generating a target polynucleotide sequence and controlling assembly of a contiguous double-stranded polynucleotide. A computer program identifies an initiating polynucleotide sequence contained in the target polynucleotide sequence, and parses the target into multiply distinct, partially complementary oligonucleotides using an algorithm based on an overlap length and oligonucleotide length.
The program generates forward and reverse-complement oligonucleotides by applying a base-by-base complement mapping for the reverse complement and printing forward and reverse oligos as parsed lists. The assembly is controlled by sequential addition on an automated work station of the partially complementary oligonucleotides synthesized on the oligonucleotide synthesizer, resulting in bi-directional extension of the initiating polynucleotide sequence.
The description further provides a computer-program and system concepts that generate oligonucleotide sets, control assembly, and support automated synthesis and ordering via server communication. System concepts include high-throughput/automation modules such as microfluidic pooling/annealing/ligation modules, high-capacity well arrays, and alternative assembly on surfaces/electrodes.
Claims Coverage
The document provides at least one independent claim and identifies a core claim set centered on computer-program generation of oligonucleotides and computer-controlled bi-directional assembly of a contiguous double-stranded polynucleotide. The inventive features focus on initiating polynucleotide identification, algorithmic parsing into partially complementary forward and reverse-complement oligonucleotides, outputting oligos to an oligonucleotide synthesizer, and controlling sequential bi-directional extension on an automated work station.
Initiating polynucleotide identification and target parsing
The program identifies an initiating polynucleotide sequence contained in the target polynucleotide sequence and parses the target polynucleotide sequence into multiply distinct, partially complementary oligonucleotides by an algorithm comprising an overlap length and generation of a reverse complemented sequence.
Forward and reverse-complement oligonucleotide generation for synthesis output
The program outputs the sequence of the multiply distinct, partially complementary oligonucleotides to an oligonucleotide synthesizer, including generating a list of parsed forward oligos and a list of parsed reverse oligos based on oligonucleotide length and overlap length.
Automated control of sequential bi-directional extension
The program controls assembly of the target polynucleotide sequence by controlling the bi-directional extension of the initiating polynucleotide sequence by the sequential addition on an automated work station of the partially complementary oligonucleotides synthesized on the oligonucleotide synthesizer resulting in production of a contiguous-double-stranded polynucleotide.
Known or previously defined target polynucleotide sequence
The target polynucleotide sequence is a known or previously defined polynucleotide sequence.
Optional uppercasing of the forward sequence with 'X' for non-ATCG bases
The program optionally converts a forward DNA sequence to upper case by iterating base-by-base and mapping a, t, c, g (and upper case forms) to their upper case equivalents and using 'X' for any other base.
The main inventive coverage is directed to a computer program that identifies an initiating polynucleotide within a known target sequence, algorithmically parses the target into partially complementary forward and reverse-complement oligonucleotides using overlap and oligonucleotide length, outputs those oligos to an oligonucleotide synthesizer, and controls assembly by sequential bi-directional extension to produce a contiguous double-stranded polynucleotide; an additional refinement specifies an optional uppercasing preprocessing step for the forward sequence with an 'X' fallback.
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