Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides.

ABSTRACT: We have improved upon the methodology and dramatically shortened the time required for accurate assembly of 5- to 6-kb segments of DNA from synthetic oligonucleotides. As a test of this methodology, we have established conditions for the rapid (14-day) assembly of the complete infectious genome of bacteriophage X174 (5386 bp) from a single pool of chemically synthesized oligonucleotides. The procedure involves three key steps: (i). gel purification of pooled oligonucleotides to reduce contamination with molecules of incorrect chain length, (ii). ligation of the oligonucleotides under stringent annealing conditions (55 degrees C) to select against annealing of molecules with incorrect sequences, and (iii). assembly of ligation products into full-length genomes by polymerase cycling assembly

SUBMITTER: Smith HO 

PROVIDER: S-EPMC307586 | biostudies-literature | 2003 Dec

REPOSITORIES: biostudies-literature