RT-PCR (PRIMER DESIGN FOR RACE)
bb1dopeg at lucano.uco.es
Mon Nov 21 11:18:24 EST 1994
According to the number of PCR-books where it is described, RACE seems
be by far the most popular method to get the sequences of the 5' and 3'
ends of a mRNA via PCR amplification of the corresponding cDNA ends.
It uses a "mix primer" consisting of two different ends, the 3' end is
oligodT (usually 17mer) and the 5' end is a "normal" oligo20mer. The
oligodT end is used to prime the synthesis of cDNA from poliadenilated
RNA, so that the oligo20mer is incorporated to the 5' end of the first
strand of the cDNA.
The cDNA end is now amplified by using an internal oligo plus the
oligo20mer as PCR primers. The amplified sequence corresponds to the 3'
end of the mRNA.
My question is: Why not to use just an oligodT for both the cDNA
and the subsequent PCR amplification?
Tm compatibility between an oligodT an any "normal" PCR primer should
be a problem since it can be adjusted by modifying the length of the
oligodT. An oligodT 30mer has a Tm as high as 68 C.
Besides, an oligodT will not form hairpins or dimers with itself and
seldomly with "normal" PCR primers, so it would not be difficult to
any preexisting internal primer.
Your comments and experiences are wellcome
Thanks in advance
Facultad de Ciencias
bv1pevir at uco.es
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