complementary DNA strand vs. ssDNA-oligoprobe hybridization
patrick.demarta at libero.it
Fri Sep 5 10:12:31 EST 2003
I have covalently bound an aminated oligoprobe of about 20bp to an aminated
microwell surface, via
homobifunctional crosslinker DSS.
I checked this conjugation by hybridizing a complementary biotinilated oligo
and detecting it using streptavidine-peroxydase complex and TMB substrate,
with good results.
The problem arise whe I try to hybridize and detect a PCR product
(the primer for the complementary strand to the capture probe being 5'
I'm aware I've to denature dsDNA before the hybridization step; I tried by
heat denaturation, 95°C for 5' and 5' on ice;
I also tried denaturation with NaOH and neutralization with Tris.
The signal is very low, near the backround.
I 'm thinking the problem is in the competition between ssDNA-probe
hybridization and renaturing of dsDNA...
my DNA fragment is 250bp and I wonder if the dsDNA form is largely
favourite rather than the hybrid with the
(respectively) small oligoprobe.
How can I favour this second reaction, despite unfavouring the first?
Using formamide in hybridization buffer can help?
Doesn't it act against the target-probe hybrids too and to a bigger extent?
Any suggestion is wellcome,
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