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Fractionating DNA Fragments Generated by Differential Display PCR

s of cDNA clones.

Differential Display PCR

figure1

DDPCR was first described by Liang and Pardee.2 In this technique (figure1), cDNA is synthesized by reverse transcription from the two RNA populations to be compared using an oligo(dT) primer to the poly(A) tail of the mRNA. PCR amplification is then performed using an anchored primer to the poly(A) tail and an arbitrary 5 primer. The PCR amplification produces DNA fragments of lengths that vary depending on the position at which the arbitrary primer bound the cDNA. The DNA fragments are then fractionated on a DNA sequencing gel, forming a fingerprint specific for the mRNA population studied. When the fingerprints of the two populations are run side by side, differences in gene expression will be indicated by the presence or absence of bands in one lane compared to the other. The differentially expressed DNA fragments may then be excised, amplified, cloned and sequenced, potentially identifying genes with increased or decreased expression in the two RNA populations. By using a number of different anchored 3 and arbitrary 5 primers in a range of combinations, RNA fingerprints can be produced that represent up to 90% of the mRNA population.3

Using the CastAway System for DDPCR

The success of the DDPCR procedure depends on the reproducibility and clarity of the sequencing gels used to compare the DNA fingerprints. Conventional sequencing, which uses manually poured gels, is labor-intensive and prone to problems, such as air bubbles and poor acrylamide polymerization. Because
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