Protocol Online logo
Top : Forum Archives: : Molecular Cloning

Primer Extension Assay versus RACE-PCR - Protocol (Aug/29/2006 )

My panelists have differing opinions on which is better to use. For those who have tried both, which would you recommend?Thanks


it depends on the system you are using. Bacteria dont undergo alternative splicing so for a bacterial gene primer extension is sufficient, in mammalian cells, there is alternative splicing and I have seen a case where primer extension was used and caused misinterpretation of the data, they figured it only went 60bp more and added that onto the known sequence by pulling sequence out of the genome, however it represented an alternatiively spliced exon which was actually much farther upstream so they mis-identified the 5'end/promoter. There are pluses and minuses of both systems, and with RACE there are alot of different techniques to use so I think it depends on what is known about your sequence, what is available to you etc.