Sticky 5'ends via ExoIII ?

the End jgraham at bronze.ucs.indiana.edu
Mon Feb 21 15:09:58 EST 1994


Yet another potentially great technique. :)

Has anyone tried the methods reported by Hsiao (NAR 21 (23) 5528-5529)
and Kaluz, Koble, and Reid (NAR 20 (16) 4369-4370) in which the blunts ends
from PCR products are converted to sticky ones by ExoIII digestion ?

The authors report that inclusion of vector homolgy into 5' primer 
ends and then treatment of either the PCR product or both the 
PCR generated insert and the vector fragment allows for directional
subcloning of PCR products. This could also be used for other variations
in which molecules which share flanking sequences devoid of restriction
sites are joined (Yang, Watson, Tucker, and Capra. 1993. NAR 21 (8) 1889-1893).

All authors appear to avoid reporting convincing statistics on the 
EFFICIENCY of such ligations and accuracy of fusion junctions. 

I am also particularly interested in the necessity for filling in gaps
introduced by ExoIII when both vector and insert are treated with
exonuclease. Apparently filling these  with T4 pol would increase
backgrounds of circularized vector.

My particular case involves inserting a randomized casette via one 
sticky end and a single base complementarity at the other end.


Thanks,


Jim
J. Graham



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