Identification of enriched sequences from a cDNA subtraction-hybridization procedure.

Abstract

The subtractive-hybridization procedure (see e.g. (1)) is a powerful procedure for obtaining cDNA populations which are enriched for upand down-regulated gene products. The method is based on the elimination of the common fragments within the cDNA populations being compared and is straight forward (1). Unfortunately, the analysis of the outcome is based on a screening hybridization assay and, hence, is indirect and time-consuming since individual molecules have to be cloned and analyzed. Another useful procedure for the isolation of specific gene products is the differential display method as developed by Liang and Pardee (2), recently optimized (3) and automated (4). In this procedure differential gene expression, in cells or tissues, is visualized by differences in the intensities of cDNA fragments after electrophoretic separation on a sequencing gel. After conversion into a cDNA using reverse transcriptase all gene products can be amplified as distinct, primer-dependent groups of cDNA by choosing a distinct oligo-dT-NN primer and a set of (short) random primers which facilitates identification (2). Although this procedure allows in principle for the identification of all mRNAs it can not be used for the isolation of underrepresented gene products. Here we report that the combination of both procedures, i.e. enriching a cDNA population by the subtractive-hybridization method (1) and identifying the enriched fragments by the differential display method (2) on a polyacrylamide gel yields a very powerful procedure that allows the identification of cDNAs that are enriched as little as 3-fold in the test fraction. In our laboratory we are studying the altered gene expression in a rat liver after a 70% hepatectomy. cDNA populations have been prepared from a normal and a regenerating (partially hepatectomized) rat liver (5). The subtractive-hybridization method as described by Wang and Brown (1), with minor modifications, was used to obtain the upand down-regulated gene products. Both cDNA populations were tagged with their own unique set of primers/adapters, CTCTTGCTTGAATTCGGACTA-3' and AATTCAGGCCAAGTCGGCCGG-3' for the normal and regenerating liver cDNA, respectively. In this way the necessity of linker removal, as described in the original procedure (1), at any point during the method was eliminated. The cDNA needed for each round of subtraction was amplified with Taq DNA polymerase (94°C for 1 min, 58°C for 90 s, and 72°C for 2 min with 30 cycles, followed by a final 5-min elongation-step at 72°C). The respective primers could not be interchanged since no product was detectable after a complete amplification cycle with a mismatched primer (not shown). In order to identify the upand down-regulated gene products after a completed subtractive-hybridization procedure, we adapted the display method from Liang and Pardee (2) with modifications. The two cDNA populations obtained after each round of subtractive-hybridization were amplified using Taq polymerase and the distinct primers in the presence of 0.5 jtM [a-S]dATP (1200 Ci/mmol) (final volume: 25 /xl). The experimental conditions were exactly the same as those used to generate the