DD AP-PCR - COMBINATION OF DIFFERENTIAL DISPLAY AND ARBITRARILY PRIMED PCR OF OLIGO(DT) CDNA/

In this report we describe the first direct comparison of differential display (DD) and arbitrarily primed PCR (AP-PGR) amplification of oli go(dT)-primed cDNA. Our results indicate that both of these widely use d RNA fingerprinting techniques have their respective advantages and l imitations. DD produces profiles specific to the anchored oligo(dT) pr imer used for cDNA synthesis. AP-PCR displays significant redundancy o f profiles generated from different oligo(dT) cDNA pools, but is not a s biased to the isolation of AT-rich or 3' sequences. It was found tha t both techniques can utilize cDNA synthesized using a generic anchore d oligo(dT) primer (dT(12)VN; equimolar amounts of dT(12)VA, dT(12)VC, dT(12)VG, and dT(12)VT, where V is dA, dC, or dG); this efficiently s elects for poly(A)(+) sequences from total RNA, and significantly redu ces the number of cDNA preparations required per experiment. Using dT( 12)VN cDNA pools generated from rat liver, spleen, and brain, the two approaches (AP-PCR and DD) were used in combination. Several known mRN As were identified; some were unique to either technique and some were common to both. Since it is the RNA which is usually the limiting res ource, maximum utilization may be achieved by generating a single pool of dT(12)VN-primed cDNA and performing both AP-PCR and DD (DD/AP-PCR) . (C) 1997 Academic Press.