Rational primer design greatly improves differential display-PCR (DD-PCR)
Rational primer design greatly improves differential display-PCR (DD-PCR)Daniel Graf*, Amanda G. Fisher and Matthias Merkenschlager
Lymphocyte Development Group, MRC Clinical Sciences Centre, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK
Received January 10, 1997;Revised and Accepted April 14, 1997
ABSTRACT
Since its conception in 1992, differential display PCR (DD-PCR) has attracted widespread interest. Theoretically an attractive cloning approach, it combines the comparative analysis of several samples with the sensitivity of PCR. Although a large number of studies embracing this technology have been initiated, few novel genes of interest have been identified, suggesting that the method has not realised its potential. The present report shows that by modifying primer design, sampling of differentially expressed genes can be greatly enhanced and relevant genes can be isolated. Using our modified conditions DD-PCR efficiently screens a wide range of gene expression levels, in which differences are represented on a linear scale.
We studied gene induction in mouse thymocytes, using exposure to antibodies against the T-cell receptor (TCR) or phorbol-12- myristate-13-acetate (PMA) and Ca++-ionophore A23187 as model systems. Thymocyte suspensions were stimulated in the presence of cycloheximide (CHX, 10 mg/ml) for 4 h with plate-bound antibody to TCR (H57, 10 [mu]g/ml adsorbed overnight) or 250 ng/ml A23187 in combination with 20 ng/ml PMA. Control cells were cultured in medium alone or in the presence of CHX. RNA was prepared by spinning the cell lysate through a CsCl cushion. cDNA was prepared with SuperscriptII (Gibco-BRL) and oligo-dT primer according to manufacturers instructions. PCR was performed with the primers indicated using [33P]dATP and hot start. The reaction mixtures were overlayered with melted paraffin, SuperTaq (HT Biotechnology, Cambridge, UK) was added and the tubes were put into a preheated thermocycler (Omnigene, Hybaid). Hot start was found to improve reproducibility. The annealing temperature for the first five cycles was 36oC, and 38oC for the subsequent 35 cycles. The PCR products were separated on 5% denaturing long ranger gels (FMC BioProduct, USA). Differential bands were cut from dried gels, reamplified and cloned into a T/A vector (1 ). Recombinant clones were screened by PCR, plasmid DNA was prepared and sequenced. All PCR reactions were run in duplicate.
Primers and cycling conditions in DD-PCR protocols were originally designed such that 100-150 different primer combinations encompass most of the estimated 15 000 expressed genes (2 ,3 ). Mitogen stimulation of mature T cells up-regulates more than 100 genes, among them CD69 and Nur77 (4 ,5 ). Assuming that mitogen stimulation is similarly effective in thymocytes one would predict on average one difference per primer combination. However, using primers and conditions as described (2 ,6 ) we encountered two types of problems. First, we observed far fewer differences than anticipated and second we isolated predominantly 10mer/10mer fragments (Table 1 ). 10mer/10mer products are in general not desirable since anchored oligo-dT primers are intended to bias the amplification to 3'-untranslated regions (3'-UTR) of genes, reducing the number of potentially amplified bands and minimising artefacts arising from variations in cDNA quality (cDNA length) or contamination with genomic DNA. Both problems can be explained by the following notions. First, the predominant amplification of 10mer/10mer products is the result of the different characteristics of random 10mer and oligo-dT primer. Because G/C interactions are stronger than those between A and T, 10mer primers with a G/C content of 50-60% are predicted to bind more strongly than the A/T-rich oligo-dT primer. As DD-PCR is a competitive reaction where primers and cDNAs are in equilibrium (see below) the amplification of 10mer/10mer products is favoured. Second, sequences in 3'-UTR often are A/T rich, making binding of random 10mer primers less likely and oligo-dT/10mer products will occur less frequently than expected.
