Differential display reverse transcription PCR (DDRT-PCR)2 was first reported by P. Liang and A.B. Paredee in 1992. The method utilized an anchor primer, including a poly(T) component with the addition of one or two select bases in reverse transcription. The first-strand cDNA was amplified via PCR using the same anchor primer and a 10-base random primer. As the DDRT-PCR method is simple and sensitive, it has become a popular technology in gene expression work. However, a major problem with the technique is its ability to produce a high rate of false-positive products. A number of approaches to overcome such problems have been suggested, including using improved gel resolution systems, performing PCR with the cDNA generated by two different reverse transcriptases, and using cytoplasmic RNA to avoid unprocessed mRNA. While there has been a focus in the literature on modification of the random primer component as a means of improving DDRT-PCR reliability, in this study we report on the impact of modifications to the traditional anchor primer component and the role of anchor primers. The addition of weight bases is important for improving the reliability of traditional anchor primer in the DDRT PCR condition and can contribute to the reproducibility of DDRT PCR products.

A simple method was developed for extracting total RNA from a single mature wheat seed embryo, which is very small and hard. Guanidine thiocyanate and chloroform were employed and the grinding of the samples was performed in a microcentrifuge tube with a plastic pestle. A "jacket" of liquid nitrogen and simplified procedures were applied to ensure the thorough grinding of the embryo tissue and to minimise the loss of samples. These measures substantially increased the recovery of total RNA in the extraction process. Reliable differential display was successfully achieved with the total RNA after DNase treatment and reverse transcription. This method makes it viable to study gene expression and gene regulation in a single wheat seed embryo. It may also give researchers the ability to analyse mRNAs in tissues or organs which were previously too small for RNA isolation using conventional procedures.

The feasibility of exploiting non-gridded bacterial artificial chromosome (BAC) libraries and some major factors affecting the efficiency of handling such libraries were studied in hexaploid wheat. Even for a bacterial culture containing only 55% recombinants, some 2000 BAC clones with inserts ranging from 45 to 245 kb could be pooled. The pooled BAC clones could be amplified by culturing for up to 6 h without losing any target clones. These results imply that even for hexaploid wheat, which has an extremely large genome, some 250 pools are sufficient for a BAC library that should satisfy many research objectives. This non-gridded strategy would dramatically reduce the cost and make robotic equipment non-essential in exploiting BAC technology. To construct a representative library and to minimise clone competition, thawing and re-freezing ligation mixtures and bacterial cultures should be avoided in BAC library construction and application.