SAGE questions

odda at my-deja.com odda at my-deja.com
Thu Nov 16 10:54:34 EST 2000


Hi Olivier,

The reason for taking two different linkers with two different primer
binding sites is that during the ditag pcr step ditags with the same
linker sequence can not be (efficiently) amplified. After the
denaturation step, instead of annealing to the primers, they can also
anneal to the second linker in the same single-stranded ditag molecule
and form a loop, but will not be amplified by Taq Polymerase.
Just check this by ligating Linkers to each other to form Linker 1 and
Linker 2 homodimers and a Linker 1 - Linker 2 heterodimer and PCR
amplify them. You will see, that only the heterodimer will be amplified.

To avoid background bands in the ditag PCR, both are not ligated to the
NlaIII cut cDNA at the same time. Since magnetic seperation is only an
enrichment and for sure will not remove all non-binding DNA, Linker
dimers could still remain in your PCR template (in the step where you
ligate the Linkers to the cDNA, also Linker-dimers could be generated,
but only homodimers and never heterodimers, since you use different
tubes).

Hope this helps.

Matthias
matthias.wahl at gsf.de


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