ANCHORED PCR

Silvia silreal at tin.it
Fri Jan 30 12:18:36 EST 2004


Hello,
I need some help. I need to know the DNA genomic sequence that follows
a short piece of sequence I already know. I though to develop an
anchored PCR protocol using restriction(only ECO RI)-legation
protocol, as in AFLP, andamplifying with both my specific primer and
primers that binds the adaptor sequence. I've got 300 bp fragment
(from agarose gel), but sequencing it I could't recognize the early
nucleotides just below my primers that I know. First problem: my
primer is designed on cDNA seq and it's rev compl works for sure (it's
not across two exons).  So I guess that the 300 bp product is not the
one I want. Even because I've tried the same process with other
pre-tested primers and at gel level I've got the same lenght band. I
haven't sequenced  those bands yet, but it seem not to be so probable
to get the same lenght band using 3 different primers while Eco RI is
supposed to cut every 1000 bp. Second problem: I have no cDNA
available.
Any help will be deeply appreciated.
Silvia Reale

Universita' del Molise
Campobasso
Italy
silreal at tin.it



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