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Fractionating DNA Fragments Generated by Differential Display PCR

Clones containing reamplified inserts have now been isolated and are presently being sequenced to identify the differentially expressed genes. All clones must be studied; in some cases, a single differentially expressed band may contain more than one DNA species.4 Once the sequences of interest have been identified, the differential expression between the two RNA populations may be confirmed using an independent technique such as Northern blot analysis or dot blotting.

Conclusions

The CastAway system is a convenient alternative to manually poured DNA sequencing gels. Because DDPCR requires reproducible, high-quality band resolution and the ability to identify bands up to 2 kb in length, using the CastAway system for DDPCR increases the likelihood of isolating coding sequence of the differentially expressed genes. Our laboratory used the CastAway system for performing DDPCR analysis of leukocytes from allergic and nonallergic blood donors.

REFERENCES

  1. Lorenz, J. (1996) Eur. Respir. Rev. 6: 218-223.

  2. Liang, P., and Pardee, A. (1992) Science 257: 967-970.

  3. Diachenko, L.B., et al. (1996) Biochem. Biophys. Res. Commun. 219: 824-828.

  4. Liang, P., and Pardee, A. (1995) Curr. Opin. Immunol. 7: 274-280.


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