PCR-BASED METHOD OF SYNTHESIZING A NUCLEIC ACID MOLECULE

摘要

There is provided a method of synthesizing a nucleic acid molecule and in one aspect, the method comprises assembling a full length template nucleic acid molecule by PCR in a PCR reaction mixture comprising a set of assembly oligonucleotides having a first average melting temperature and a set of outer amplification primers having a second average melting temperature that is lower than the first average melting temperature, wherein said assembling comprises subjecting the PCR reaction mixture to a first annealing temperature that is higher than the second average melting temperature and; amplifying the full length template nucleic acid molecule by PCR in the PCR reaction mixture wherein said amplifying comprises subjecting the PCR reaction mixture to a second annealing temperature that permits annealing of the outer amplification primers to the full length template nucleic acid molecule.