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Fractionating DNA Fragments Generated by Differential Display PCR

CastAway system application

Iain Kilty Phil Vickers
Pfizer Central Research, Sandwich, UK. 01304 618482

Differential display PCR (DDPCR) is a widely used technique for identifying genes differentially expressed between cell types. Application of DDPCR to allergic disease provides a potential means for elucidating the mechanisms involved in allergic responses. In order to successfully apply DDPCR, high-quality, reproducible denaturing polyacrylamide gels are required to fractionate PCR fragments. Stratagenes CastAway system* is convenient and consistently yields the high-quality band resolution that is necessary for DDPCR.

Allergic disease represents a major public health problem in most countries, affecting people of all races. Over 100 million people worldwide suffer from asthma alone.1 Allergic disease is treated predominantly with the use of steroids. Although steroids are effective in eliminating the symptoms of allergic disease, they also cause a number of detrimental side effects. Thus, a greater understanding of the genes involved in the pathogenesis of allergic disease is needed to allow more acceptable drugs to be developed.

As leukocytes play a key role in mediating allergic responses, a comparison of the expression levels of genes in the leukocytes of people suffering from allergic disease as compared to nonallergic controls may provide insights into the basis of such disease. A number of methods may be used for identifying differentially expressed genes, such as high-throughput sequencing of expressed sequence tags (ESTs), subtractive hybridization of cDNA and DDPCR. DDPCR is often the method of choice for identifying differentially expressed genes because it (1) requires relatively little starting material, (2) can be used to screen a large proportion of the mRNA population and (3) does not require the sequencing of large number
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