Methods for Nucleic Acid Assembly and High Throughput Sequencing

摘要

Methods and apparatus of some aspects of the invention relate to the synthesis of high fidelity polynucleotides. In particular, aspects of the invention relate to concurrent enzymatic removal of amplification sequences and ligation of processed oligonucleotides into nucleic acid assemblies. According to some embodiments, the invention provides a method for producing a target nucleic acid having a predefined sequence. In some embodiments, the method comprises the step of providing a plurality of oligonucleotides, wherein each oligonucleotides comprises (i) an internal sequence identical to a different portion of a sequence of a target nucleic acid, (ii) a 5′ sequence flanking the 5′ end of the internal sequence and a 3′ flanking sequence flanking the 3′ end of the internal sequence, each of the flanking sequence comprising a primer recognition site for a primer pair and a restriction enzyme recognition site.

主权项

1. A method of producing a target nucleic acid having a predefined sequence, the method comprising:
a) providing a first mixture comprising:
(i) a first pool of oligonucleotides comprising a first plurality of oligonucleotides comprising a sequence identical to the 5′ end of the target nucleic acid, a second plurality of oligonucleotides comprising a sequence identical to the 3′ end of the target nucleic acid, and a plurality of oligonucleotides comprising a sequence identical to a different portion of a sequence of a target nucleic acid, each of the oligonucleotides having an overlapping sequence region corresponding to a sequence region in a next oligonucleotide, the oligonucleotides in the first pool together comprising the target nucleic acid sequence;(ii) a restriction enzyme, and b) exposing the first mixture to a ligase and subjecting the first mixture comprising the ligase to conditions suitable to promote restriction enzyme digestion and ligation, thereby generating the target nucleic acid.