Compositions, methods and apparatus for oligonucleotides synthesis
Inventors
Hall, Giles F. • Lawton, Scott S.
Assignees
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Publication Number
US-11242523-B2
Publication Date
2022-02-08
Expiration Date
Abstract
Aspects of the invention relate to methods, compositions for synthesizing oligonucleotides having a predefined sequence.
Core Innovation
The invention relates to generating a target oligonucleotide having a predefined sequence by designing at least one plurality of oligonucleotides with an identical predefined internal sequence, but with different oligonucleotide lengths. Each oligonucleotide includes a 3′ flanking sequence at the 3′ end of the internal sequence and a 5′ flanking sequence at the 5′ end of the internal sequence, and the 3′ flanking sequence comprises primer recognition site, a restriction enzyme recognition site, and a padding nucleotide sequence of a different length.
The invention further implements synthesizing the designed plurality on a solid support and isolating the target oligonucleotide having the predefined sequence. The disclosed approach includes forming a redundant array in which the redundant array comprises redundant internal sequences, and the oligonucleotides in the array include identical predefined internal sequences while having different lengths via differently sized padding nucleotide sequences.
In addition, the invention provides a composition for assembly of a target nucleic acid having a predefined sequence, where a first plurality of oligonucleotides comprises a first internal sequence identical to the 5′ end of the target nucleic acid and a second plurality comprises a second internal sequence identical to the 3′ end. One or more optional pluralities may include internal sequences identical to different portions of the target nucleic acid, where oligonucleotides overlap by having sequence regions overlapping with sequence regions in other pluralities.
Claims Coverage
The partial content includes three independent claims (clm-00001, clm-00012, clm-00016). Across these claims, the inventive features center on identical predefined internal sequences across oligonucleotide variants of different lengths, with padding nucleotide sequences of different lengths in defined 3′ and/or flanking regions that include primer recognition and restriction enzyme recognition sites, together with overlap and assembly relationships for reconstructing a target nucleic acid sequence.
Different-length oligonucleotide variants sharing an identical internal sequence with defined flanks
Designing at least one plurality of oligonucleotides, each oligonucleotide having a different length while each having an identical predefined internal sequence, a 3′ flanking sequence at the 3′ end of the internal sequence, and a 5′ flanking sequence at the 5′ end of the internal sequence, wherein the 3′ flanking sequence comprises a primer recognition site, a restriction enzyme recognition site, and a padding nucleotide sequence of a different length.
Solid support synthesis and isolating a target oligonucleotide with the predefined sequence
Synthesizing the at least one plurality of oligonucleotides on a solid support and isolating the target oligonucleotide having the predefined sequence.
Redundant array with different-length padding nucleotides and redundant internal sequences
A redundant array comprising at least one plurality of oligonucleotide sequences, each oligonucleotide of each plurality having a different length while each has an identical predefined internal sequence, a 3′ flanking sequence at the 3′ end of the internal sequence and a 5′ flanking sequence at the 5′ end of the internal sequence, where the 3′ flanking sequence comprises a padding nucleotide sequence with a different length, where the 5′ flanking sequence comprises a primer recognition site and a restriction enzyme recognition site, and wherein the redundant array comprises redundant internal sequences.
Overlapping-plurality oligonucleotide composition for assembly of a target nucleic acid
A composition for the assembly of a target nucleic acid having a predefined sequence comprising a first plurality of oligonucleotides comprising a first internal sequence identical to the 5′ end of the target nucleic acid; a second plurality of oligonucleotides comprising a second internal sequence identical to the 3′ end of the target nucleic acid; and optionally one or more pluralities of oligonucleotides each comprising an internal sequence identical to a different portion of the sequence of the target nucleic acid, wherein each oligonucleotide of each plurality has a sequence region overlapping with a sequence region in each oligonucleotide of another plurality.
3′ flanking padding nucleotide sequences with different lengths in an assembly composition
Each oligonucleotide has 5′ and 3′ flanking sequences at the 5′ end and the 3′ end of each internal sequence, each flanking sequence comprising a primer recognition site and a restriction enzyme recognition site, and each 3′ flanking sequence further comprises a padding nucleotide sequence that has a different length than other padding sequences within each plurality of oligonucleotides.
Across the independent claims, the shared inventive concept is constructing oligonucleotide sets, arrays, or assembly compositions from oligonucleotides that share identical internal sequences but differ in length by varying padding nucleotide sequences, with flanking regions that include primer recognition and restriction enzyme recognition sites, enabling synthesis and isolation of a target sequence and enabling assembly by overlapping oligonucleotide regions.
Stated Advantages
Documented Applications
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