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Fractionating DNA Fragments Generated by Differential Display PCR

CastAway gels are provided polymerized, the time required to prepare the denaturing polyacrylamide gel for fractionating the DNA fingerprint is dramatically reduced. Moreover, the CastAway precast gels are only 0.25-mm thick, allowing them to be run faster than conventional gels and improving band resolution. With the thin nature of the CastAway gels, bands up to 2 kb in length can be resolved. Increasing the size of the DNA fragments that can be identified is of particular importance to DDPCR as the DNA fragments are biased toward the 3 end of the initial mRNA due to the anchored 3 primer. Larger DNA fragments are more likely to contain coding sequence for the differentially expressed gene of interest, whereas DNA fragments of less than 500 bp may contain only 3-untranslated sequences, making identification of the gene difficult.

DDPCR: Allergic and Nonallergic Blood Donors

figure 2

Using the CastAway system, we identified 10 differentially expressed bands with increased expression in the peripheral blood leukocytes of donors suffering from allergic disease compared to control donors. The sizes of these differentially expressed fragments ranged between approximately 0.5 and 1.5 kb. An autoradiograph from one DDPCR experiment is shown (figure 2). Arrows indicate the positions of two differentially expressed bands that were identified using this particular primer pair. Each of the differentially expressed bands was excised from the gel, which had been dried on a glass plate. The DNA from the excised gel slices was reamplified and cloned into a suitable vector. Of the 10 differentially expressed bands identified, 7 were successfully reamplified and cloned.
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