METHOD FOR ASSEMBLY OF POLYNUCLEIC ACID SEQUENCES

• Main Authors: KNIGHT, Tom, CHE, Austin, KELLY, Jason, CANTON, Barry, SHETTY, Reshma
• Format: Patent
• Language: English; French; German
• Published: 23.08.2017
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS THEREOF; CULTURE MEDIA; ENZYMOLOGY; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; VINEGAR; WINE

A method for the assembly of a polynucleic acid sequence from a plurality of nucleic acid sequences in which the polynucleic acid sequence is of a formula N n+1 , in which N represents a nucleic acid sequence and where n is 1 or greater than 1 and each N may be the same or a different nucleic acid sequence, in which the method comprises: (i) providing a first nucleic acid sequence N1 in a vector, using a restriction enzyme to cut the nucleic acid sequence out from the vector and ligating an oligonucleotide linker sequence L1 3' to the 3'-end of the nucleic acid sequence; (ii) providing a second nucleic acid sequence N2 in a vector, using a restriction enzyme to cut the nucleic acid sequence out from the vector and ligating an oligonucleotide linker sequence L2 5' to the 5'-end of the nucleic acid sequence, wherein the 5'-end linker sequence L2 5' of nucleic acid sequence N2 is complementary to the 3'-end linker sequence L1 3' of nucleic acid sequence N1; and ligating an oligonucleotide linker sequence L2 3' to the 3'-end of the nucleic acid sequence N2; and (iii) providing one or more additional nucleic acid sequence(s) N in one or more vector(s), using a restriction enzyme to cut the nucleic acid sequence(s) out from the vector(s), wherein nucleic acid sequence N2 has an oligonucleotide linker sequence L2 3' at the 3'-end of the nucleic acid sequence, and wherein said one or more additional nucleic acid sequences N comprises a terminal additional nucleic acid sequence NZ, and ligating an oligonucleotide linker sequence to the 3'-end of each additional nucleic acid sequence N, wherein said terminal additional nucleic acid sequence NZ optionally lacks an oligonucleotide linker sequence at its 3'-end, and ligating an oligonucleotide linker sequence to the 5'-end of each additional nucleic acid sequence N, wherein for the first additional nucleic acid sequence N3 the 5'-end linker sequence L3 5' is complementary to the 3'-end linker sequence L2 3' of nucleic acid sequence N2 and for each second and subsequent additional nucleic acid sequence N the 5'-end linker sequence is complementary to the 3'-end linker sequence of the respective preceding additional nucleic acid sequence.