David F. Mark

Quantitation of mRNA by the Polymerase Chain Reaction

A method for the quantitation of specific mRNA species by the polymerase chain reaction (PCR) has been developed by using a synthetic RNA as an internal standard. The specific target mRNA and the internal standard are coamplified in one reaction in which the same primers are used. The amount of mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The synthetic internal standard RNA consists of a linear array of the sequences of upstream primers of multiple target genes followed by the complementary sequences to their downstream primers in the same order. This quantitative PCR method provides a rapid and reliable way to quantify the amount of a specific mRNA in a sample of less than 0.1 ng of total RNA. In addition, the same internal standard RNA is used, with appropriate primer pairs, to quantitate multiple different mRNA species.

Site-specific mutagenesis to modify the human tumor necrosis factor gene.

Publisher Summary The development of oligonucleotide-directed site-specific mutagenesis on single-stranded phage template has provided a powerful new technique in the study of structure-function relationships of proteins. This chapter describes a simplified procedure for oligonucleotide-directed site-specific mutagenesis on M13 single-stranded phage deoxyribo nucleic acid (DNA) templates. The rationale behind this approach is to make use of the DNA repair mechanism present in the E. coli host to repair the gapped M13 phage DNA into the covalently closed circular form before replication of the DNA molecules. The mutagenesis procedure described in this chapter provides a rapid method for the modification of cloned genes. It takes advantage of the ability of E. coli to efficiently repair gapped circular DNA molecules, to shorten the overall time of the mutagenesis, from initiation of the mutagenesis reaction to the identification of the mutant phage plaque. Although this faster method is less efficient in the conversion of parent DNA to mutant DNA molecules, the ability to use the same oligonucleotide primer as a probe to identify the mutant phage plaques effectively compensates for this inefficiency.

High-throughput measurements of biochemical responses using the plate::vision multimode 96 minilens array reader.

The plate::vision is a high-throughput multimode reader capable of reading absorbance, fluorescence, fluorescence polarization, time-resolved fluorescence, and luminescence. Its performance has been shown to be quite comparable with other readers. When the reader is integrated into the plate::explorer, an ultrahigh-throughput screening system with event-driven software and parallel plate-handling devices, it becomes possible to run complicated assays with kinetic readouts in high-density microtiter plate formats for high-throughput screening. For the past 5 years, we have used the plate::vision and the plate::explorer to run screens and have generated more than 30 million data points. Their throughput, performance, and robustness have speeded up our drug discovery process greatly.