Methods and devices for nucleic acids synthesis
Inventors
Jacobson, Joseph • Kung, Li-yun A. • Wilson, Andrew Kirk • Ramu, Senthil • SCHINDLER, DANIEL • Hudson, Michael
Assignees
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Publication Number
US-11084014-B2
Publication Date
2021-08-10
Expiration Date
Abstract
Methods and apparatus relate to the synthesis of polynucleotides having a predefined sequence on a support. Assembly methods include primer extension to generate overlapping construction oligonucleotides and assembly of the polynucleotides of interest onto an anchor support-bound oligonucleotides. Methods and apparatus for selection of polynucleotides having the predefined sequence and/or length are disclosed.
Core Innovation
The invention provides methods for generating a polynucleotide having a predefined sequence by using support-bound double-stranded polynucleotides on a solid support, including microarrays. The support-bound double-stranded polynucleotides comprise one or more construction oligonucleotides and a free single-stranded overhang, where the single-stranded overhang comprises the sequence of a terminal construction oligonucleotide N.
A stem-loop oligonucleotide is provided with a single-stranded overhang complementary to the sequence of the terminal construction oligonucleotide N, the stem-loop is hybridized to the free overhang, and the stem-loop is ligated to the free overhang. A selection and screening strategy is used to remove undesired products by degrading polynucleotide sequences using an exonuclease, thereby enriching for correct ligation products that contain the terminal construction oligonucleotide N.
Additional refinements include generating construction oligonucleotides on support features by primer extension and using hybridization and ligation on immobilized support-bound oligonucleotides to assemble predefined polynucleotide sequences with high fidelity. Error correction and product selection features based on mismatch recognition and polynucleotide length selection are also described.
Claims Coverage
The claim coverage identifies one independent method claim directed to generating a predefined-sequence polynucleotide on a solid support using hybridization and ligation with a stem-loop oligonucleotide, followed by exonuclease-based degradation of non-matching end-containing products. The independent claim is refined by dependent claims that add support synthesis, release, exonuclease specificity, stem-loop restriction-site features, and amplification.
Support-bound double-stranded polynucleotides with terminal overhang sequence N
Synthesizing a plurality of support-bound double-stranded polynucleotides comprising one or more construction oligonucleotides and a free single-stranded overhang, where the plurality comprises the polynucleotide having the predefined sequence and the free overhang comprises the sequence of a terminal construction oligonucleotide N.
Stem-loop oligonucleotide hybridization to complementary free overhang
Providing a stem-loop oligonucleotide comprising a single-stranded overhang complementary to the sequence of the terminal construction oligonucleotide N, and hybridizing the stem-loop oligonucleotide to the free overhang of the polynucleotide having the predefined sequence.
Ligation of stem-loop oligonucleotide to free overhang
Ligating the stem-loop oligonucleotide to the free overhang of the polynucleotide having the predefined sequence.
Exonuclease degradation of non-terminal construction overhang products
Degrading, using an exonuclease, polynucleotide sequences that comprise a free 3′ or 5′ end and that do not comprise the terminal construction oligonucleotide N.
Support synthesis by polymerase chain extension
Synthesizing the plurality of support-bound double-stranded polynucleotides on a support by polymerase chain extension.
Polynucleotide release by Type II restriction enzyme or UDG/Endonuclease VIII mixture
Releasing the polynucleotides using a Type II restriction enzyme or using a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease VIII.
Single strand-specific exonuclease selection at 3′ or 5′ ends
Using an exonuclease that is either a single strand-specific 3′ exonuclease or a single strand-specific 5′ exonuclease.
Stem-loop oligonucleotide including a type II restriction site
Characterizing the stem-loop oligonucleotide so that it includes a type II restriction site.
Amplification of the predefined polynucleotide
Further including amplifying a predefined polynucleotide with a predefined sequence.
Overall, the claim set covers assembling a predefined-sequence polynucleotide by support-bound overhang-directed hybridization and ligation using a stem-loop oligonucleotide, while enforcing fidelity by exonuclease degradation of polynucleotide sequences that have free 3′ or 5′ ends but lack the terminal construction oligonucleotide N. Dependent claims narrow features by specifying support synthesis, release reagents, exonuclease end specificity, stem-loop inclusion of a type II restriction site, and an optional amplification step.
Stated Advantages
High-fidelity polynucleotide assembly of predefined sequence.
Removal of undesired products by degrading polynucleotide sequences that comprise a free 3′ or 5′ end and do not comprise the terminal construction oligonucleotide N.
Error correction using mismatch-recognizing nucleases such as CEL I.
Polynucleotide length selection using a molecular ruler based on anchor spacing.
Documented Applications
Generation of predefined-sequence polynucleotides using solid support features including microarrays.
Sequential or parallel transfer of construction oligonucleotides between arrays in fluid media with diffusion control and ligase-mediated capture.
Automated system control for polynucleotide assembly using the described features.
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