| abstract |
The present invention provides 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 Nn+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 which has an oligonucleotide linker sequence L1 3' at the 3'-end of the nucleic acid sequence; (ii) providing a second nucleic acid sequence N2 which optionally has an oligonucleotide linker sequence L2 3 at the 3'-end of the nucleic acid sequence and which has an oligonucleotide linker sequence L2 5 at 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 L13 of nucleic acid sequence N1; (iii) optionally providing one or more additional nucleic acid sequences N, 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 wherein each additional nucleic acid sequence N has an oligonucleotide linker sequence at its 3'-end, wherein said terminal additional nucleic acid sequence NZ optionally lacks an oligonucleotide linker sequence at its 3'-end and wherein each additional nucleic acid sequence N has an oligonucleotide linker sequence at its 5'-end, 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; and (iv) ligating said nucleic acid sequences to form said polynucleic acid sequence; wherein at least step (iv) is carried out on a microfluidic device. |