When thinking about making "giant" oligos one should remember that
chemical synthesis is not as good as Nature in that mistakes (base modifications) will be present in any synthetic oligo ie a small %age will carry eg
deaminations, T-C modifications etc. Not normally a problem with shorter
oligos but let us assume a worst case of 1 in 50 nucleotides being modified
in some deleterious way. If you make a 200 mer (presumably your synthesizer
is in a very dry place like the Nevada desert) then most of them will carry
some modification and could screw things up.
If you want a specific sequence at the end of the day you might just try
the standard shotgun Klenow approach with eg 50-70 mers, thus:
NNNNNNNNNNNNNNN(5'P)-NN(50 orso)NNNNNNNNNN (5'P)NNNNNNNNNNNNNNN
3'OH-NNNNNNNNNNNN(5'-P) 3'OHNNNNNNN(5'P) NNNNNNN
The lower oligos are ca 15 nucleotides, the upper 50-70. The bottom
strand is produced using 5'-phosphorylated oligos and eg T7 polymerase.
You can incorporate res. sites etc at the ends for cloning.
The PCR techniques may be dandy but whatch out for artifacts introduced
by multiple cycles. The above method needs only one cycle.
Cheers
Jon R Sayers (Cell and Molecular Biology), Biochemistry Dept.,
University College North Wales, Univ of Wales, Bangor, LL57 2UW.
Tel +44 248 38 23 54
Fax +44 248 370731
Email BSS032 at BANGOR.AC.UK
