Enhanced cloning efficiency of differential display PCR amplicons

Abstract

Differential display offers a rapid and simultaneous comparison of expression profiles of mRNA from variant cells having the same genetic background. An inherent flaw of the technique may yield false-positives on subsequent analyses. The only source of the band of interest is the denaturing sequencing gel from which the band is identified. Consequently, the integrity of the fragment is compromised, and cloning (required to gain sequence information) becomes costly and time-consuming. Described herein are modifications used to stabilize the integrity of the fragment, as well as to increase the number of transcripts cloned. Additional steps were added to the processing of the differential display reverse transcription-polymerase chain reaction (DDRT-PCR) fragment, and cloning reactions were either adapted to concentrate the product or specifically optimized at certain points to prompt efficiency. Therefore, the overall cost and time spent analyzing truepositives, as confirmed by reverse Northern blots, was reduced.