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The average length of an mRNA molecule is 1.2 kb and the size limit that can be resolved on an acrylamide gel is 500bp, so ideally the 5 ' primer should have a specificity such that it will anneal fairly frequently. This will allow the amplification of cDNA fragments of varying sizes fi*om single mRNA species. A PCR primer 10 base pairs in length was found to produce 50-100 products fi"om a single cDNA sample which is adequate for resolution in an acrylamide gel (Bauer et a l, 1993, Liang et a l, 1992b).

Although shorter primers would be expected to anneal more fi-equently than 10- mers, the annealing temperature required for the PCR reaction is usually too low for efficient binding. It has been found that the 10-mer primers bind with some degeneracy to the cDNA sequences (Bauer et a l, 1993, Liang et a l, 1992b). Up to four mismatches can be tolerated if they are clustered at the 5' end of the primer, but two internal

mismatches will fail to detect a known sequence. This means that in some ways the 10- mer is acting like a 6- or 7-mer and will therefore bind more frequently than would be hypothesized. This was shown to be correct. Assuming there are 15 000 different mRNA species in a cell, and that only those cDNA smaller than SOObp can be visualized on an acrylamide gel, then a 10-mer primer would only be expected to prime and the cDNAs successfully amplified and visualized from one or less mRNAs (this is based on the assumption that a 10-mer primer will bind on average every 1049bp) (Liang et a l,

1992b). Experimentally, however, between 50 and 100 cDNAs are displayed on a gel; this would suggest that the assumption that the 10-mers are acting in a degenerate fashion is correct (Liang et a l, 1992).

3.4.3 Choice o f3 arb itrary 10-mer primers

Different investigators have designed different 10-mers but they are usually 50% GC rich and are checked to be non-palindromic so that they will not self-anneal or form stem-loop structures. It has been shown that arbitrary primers with GC pairs at the 5' end are superior to those with GC pairs at the 3 ' end, and that increasing the number of G or C residues at the 3 ' end increases the amplification (Mou et a l, 1994).

3.4.4 Amplifying the total mRNA population of a cell

It has been suggested that if twelve anchor primers o f the form T^M N (where M and N represent different bases o f which M is never T) and twenty-six arbitrary 10-mer primers are used in 312 PCR reactions that theoretically all o f the 15 000 mRNAs present in a cell should be amplified (Bauer et a l, 1993). This should also be true when using the four anchor primers of the form T12VN (where V is degenerate and N represents four different bases) in (26 x 4) PCR reactions.

This assumption will be most important for researchers trying to identify exhaustively all the differentially expressed genes between several cell populations.

3.5 Applications of Differential Display RT-PCR

Genes are differentially expressed both during normal development and during the progression of disease. Instances o f both cases have been studied in the past few years as the technique has been developed, refined and optimized. The technique can also

products can then be used as probes on Northern or Southern blots and used to isolate corresponding sequences from genomic or cDNA libraries.

3.6 Variety of investigations using Differential Display RT-PCR

Below is a description o f just some o f the experiments that have been carried out using Differential Display, demonstrating the versatility of the technique and the variety of investigations for which the technique has been used. These experiments also highlight some o f the limits to which DDRT can be taken.

3.6.1 Complex tissues - tum ours

DDRT is ideal for analysis o f cancer: a pathological process involving altered gene expression in specific tissues. A study that highlights the potential of DDRT for identifying genes involved in causing cancer was carried out by Sager et a l in 1993. In this case a comparison o f two cell lines from a single patient, primary mammary tumour and metastatic tumour, was undertaken. cDNAs expressed specifically in cancerous cells and not in normal cells could be possible candidates for oncogenes, whereas those expressed in normal cells but not in tumour cells are candidates for tumour suppressor genes.

One cDNA fragment that was differentially expressed was partially sequenced and identified as alpha 6 integrin. When used as a probe on a Northern blot containing RNA from a range of normal and tumour derived cells, it was found that this previously identified gene was expressed most strongly in normal cells, less in primary tumour cells and only faintly in metastatic cells. This confirmed that the gene was down-regulated rather than mutated in cancerous cells and is thus a candidate for a tumour suppressor gene.

Liang et a l (1992b) have also carried out experiments comparing mRNAs from normal and tumour-derived human mammary epithelial cells, cultured under the same conditions. Three candidate cDNAs were isolated in this case, one of which was analysed further and shown to be novel, its differential expression being confirmed by hybridizations to a Northern blot containing the same RNA as used for the DDRT reaction.

3.6.2 Differing physiological conditions

DDRT also lends itself to the study of altered gene expression in response to differing physiological conditions, whether they be during the normal course of cell development or as part o f a disease process. Several genes were isolated by Aiello et al.

(1994) whilst studying the regulation of gene expression in retinal pericytes exposed to varying concentrations o f glucose. Hyperglycaemia causes retinopathy in many patients with diabetes and it was assumed that alterations in gene expression was causing irreversible effects. Cells were cultured under normal (5.5mM) and pathologic (20mM) glucose concentrations, and after performing DDRT on mRNA isolated from both cultures, 35 candidate clones were isolated o f which 25 were found to be distinct genes. Altered gene expression was confirmed by Northerns for 10 o f these, with undetectable signals for 12 and non-regulation found for 3. The expression o f only one of these genes had been previously reported to be altered by glucose (fibronectin).

The technique was also used to identify genes causing chronic cardiac rejection in rats (Utans et a l, 1994). This occurs after allogeneic (Lewis rat to F344 rat) but not syngeneic (Lewis to Lewis) cardiac transplantation, and results in transplant arteriosclerosis. RNA from four allogeneic hearts showing signs o f rejection were compared with RNA from two histologically normal hearts that had been subjected to syngeneic transplantation. After DDRT was carried out, 12 cDNA candidates were isolated and 5 were confirmed to be upregulated over time in hearts undergoing chronic rejection. Three of these represented known genes not previously associated with chronic rejection and the other two were novel genes.

Bauer et a l (1993) used the technique to analyse differential gene expression in the regenerating mouse liver. Mouse liver was compared before and after hepatectomy at 3 time points of regeneration and with the resting liver. Twelve anchor primers of the form T12VN and twenty-six arbitrary 10-mer primers were used in 312 PCR reactions that should theoretically amplify cDNAs from the approximately 15 000 mRNAs present in each cell. 38 004 cDNAs were amplified in total o f which 70 were found to be differentially expressed between the four stages of liver regeneration.