Synthetic oligo with "blocked" 3'-OH group
Cheung C. Yue
ccy at po.CWRU.Edu
Wed Dec 16 12:06:28 EST 1992
Seems like a crazy idea, but the paper on "ligation-anchored PCR" in
PNAS 89:9823, strongly suggests the utility of such 3'-blocked oligos.
In that paper, the 5'-RACE procedure is now an anchor oligo ligation
procedure. Briefly, cDNA is primed by a sequence-specific oligo, and
ligated to the anchor oligo which is 5'-phosphorylated and 3'-blocked
(by tailing with dideoxynucleotide) using RNA ligase. This ligated
species can then be amplified by PCR using sequence-specific oligo and
an oligo complementary to the anchor. The absence of 5' phosphate and
presence of 3'OH on the sequence-specific oligo, and the reverse for
the anchor oligo ensures a one-way ligation.
Real neat method. I was just thinking that if in the synthesis of the
anchor oligo, one could chemically knock off the 3'-OH while cleaving
the oligo off the column, then one could avoid the tailing reaction which
is obligatory in the current version of the procedure. Not being a DNA
synthesis chemist, I don't know how one might do that (without screwing
up the rest of the molecule). Any suggestions?
ccy at po.cwru.edu
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