Alice M. Wang

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.

Detection of Gene Expression

The detection of gene expression can be accomplished in several ways. “Classical” methods (still important, of course) relied on the observation of biochemical or phenotypic changes in cells or organisms in order to determine the expression of a specific gene. Later, advances in macromolecular separation technology made feasible the identification and isolation of a particular gene product or protein molecule. With the advent of recombinant DNA techniques, it is now possible to detect and analyze the transcriptional product of any gene. There are several methods now in widespread use for studying specific RNA molecules. These methods include in situ hybridization,1 Northern gels,2 dot-or slot-blots,2,3 S-1 nuclease assays,4 and RNase protection studies.5 In this chapter, we describe a new and powerful method to detect gene expression at the RNA level. Protocols will be outlined and discussed in detail and some applications will be briefly described. No attempt has been made to exhaustively review the literature in this fast moving area, so we wish to apologize beforehand to the numerous authors whose work may have not been cited in this chapter.

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.