METHOD FOR THE DIFFERENTIAL SCREENING OF GENE EXPRESSION BY RANDOM PRIMED REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION

摘要

A process for the differential screening of gene expression in a biological sample by RP RT-PCR, wherein the PCR is effected using a plurality of oligonucleotides, the sequences of which have been determined by a process comprising: (I) generation of random primer sequences having a CG:AT ratio of 2:1, no stop codon, not more than three consecutive identical nucleotides and no palindromic 5'- and 3'-ends: (II) screening of the primer sequences generated in (I) by simulation of PCR on non-redundant mammalian nucleotide databank entries having a coding region of at least 1000 bp. and calculating for each primer sequence: (i) the efficiency index, which is the ratio of the number of PCR products comprising coding sequences using said primer sequence to the modal number of PCR products comprising coding sequences obtained for each of the whole set of tested primers generated in (I); (ii) selectivity index, which is the ratio between the probability of yielding a PCR product comprising coding sequences to that of yielding a PCR product comprising sequences encoding 3'-untranslated regions; (III) selection of primer sequences, screened as in (II), according to the said indices for use in PCR. The oligonucleotide primers preferably consist of eight C or G and four A or T, and each primer may differ from other primers in at least five out of eight bases at the 3'-end. The simulated PCR may be effected on non-redundant human, or mouse, databanks from which variable regions of immunoglobulins, T-cell receptors and intron regions are eliminated. The oligonucleotide primers preferably have an efficiency index of between 2 and 10 and a selectivity index of greater than 1, and may be partially degenerate in the final position at the 3'-end.