Mammalian polyadenylation sites: implications for differential display.

Differential display relies on a series of anchored primers to divide the total mRNA population into subsets of roughly equal size. However, this will only occur if the dinucleotide targeted by the anchor region of the anchored primers has a random frequency distribution [i.e. each of the 12 possible dinucleotides preceding the poly(A) tail occur with the same frequency]. Previous reports have suggested that this is not the case and that the frequency distribution of the targeted dinucleotide can vary as much as 10-fold. In an analysis of several hundred unrelated mammalian mRNA sequences, we confirmed that the frequency of this particular dinucleotide does vary, although <3-fold. Of equal importance, however, we found that the number of bands displayed with each of the respective anchored primers was not affected by these variations in dinucleotide frequency, suggesting that anchored primer promiscuity permits mispriming during the reverse transcription stage of differential display. Close examination of this issue suggested that both mispriming at the anchor region and internal mispriming are common in differential display reverse transcription and implies that repetitive sampling occurs exten-sively in differential display. Thus, reverse transcriptase mispriming may considerably reduce the efficiency of differential display.