Global analysis of gene expression by differential display: a mathematical model, in Methods in Molecular Biology
Differential Display Methods and Protocols, pp.
October (4th Quarter/Autumn)
Differential display (DD) is one of the most commonly used approaches for identifying differentially expressed genes. However, there has been lack of an accurate guidance on how many DD polymerase chain reaction (PCR) primer combinations are needed to display most of the genes expressed in a eukaryotic cell. This study critically evaluated the gene coverage by DD as a function of the number of arbitrary primers, the number of 3′ bases of an arbitrary primer required to completely match an mRNA target sequence, the additional 5′ base match(s) of arbitrary primers in first-strand cDNA recognition, and the length of mRNA tails being analyzed. The resulting new DD mathematical model predicts that 80–160 arbitrary 13mers, when used in combinations with three one-base anchored oligo-dT primers, would allow any given mRNA within a eukaryotic cell to be detected with a 74–93% probability, respectively. The prediction was supported by both computer simulation of the DD process and experimental data from a comprehensive fluorescent DD screening for target genes of tumor-suppressor p53. Thus, this work provides a theoretical foundation upon which global analysis of gene expression by DD can be pursued.