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Giant oligos

bss032 at uk.ac.bangor.vaxa bss032 at uk.ac.bangor.vaxa
Fri Aug 13 09:03:37 EST 1993


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



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