( CastAway system application Iain K...)Fractionating DNA Fragments Generated by Differential Display PCR

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 numbers 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 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. 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.