Our lab has had difficulties building genes from
complementary 40-60 nt oligonucleotides generated by the
Applied Biosystems 392 DNA/RNA synthesizer. A typical
gene construction involves annealing 6-10 oligos,
ligation, cloning into a sequencing vector, and then
sequencing the products. The problem is that the
resultant genes often carry one or more mutations. These
mutations appear to be randomly distributed and are not
concentrated around the junctions between the oligos.
The source of these mutations is not operator error or
errors in designing the oligos because in a bank of
clones coding for gene X, a given mutation will only
occur once while the rest of the clones are correct.
Transition, transversion and deletion mutations have been
Has anyone had any similar experiences?
Does anyone know what is causing the mutations?
a) Probabilistic errors in the oligo synthesis.
b) UV exposure mutations (during UV shadowing).
c) Secondary structure of the oligos.
Thank you in advance