Since its introduction nearly 30 years ago, PCR has become a fundamental laboratory technique in molecular biology. Initially requiring only a few reagents, a source of heat, and time for manual thermocycling, PCR technology has since spurred an industry focused on automating and improving thermocycling, developing instrumentation for real-time quantitative monitoring of amplification reactions, and enhancing the properties of polymerases.
With this in mind, the editors at BioTechniques identified our favorite PCR methods published in 2012. From a new RT-PCR method for strand-specific quantification of gene expression to a PCR-based screening approach for more rapidly identifying bacterial virulence genes, these methods show the power and progress of PCR.
Lin Feng, Susanna Lintula, Tho Huu Ho, Maria Anastasina, Annukka Paju, Caj Haglund, Ulf-Håkan Stenman, Kristina Hotakainen, Arto Orpana, Denis Kainov, and Jakob Stenman,BioTechniques, Vol. 52, No. 4, April 2012, pp. 263–270
Although we have known for years that gene expression is controlled through DNA structure and sets of proteins capable of binding regulatory sequences, recent studies have also demonstrated the importance of antisense transcription and non-coding RNAs in gene regulation. In fact, techniques such as RNA-seq are improving our basic understanding of the wide variety of RNA transcripts present in cells. These findings have led to a growing interest in understanding bidirectional transcription and compensating for this when measuring strand-specific transcript levels in cells. With its sensitivity, RT-PCR is particularly attractive for detecting nucleic acids present at low copy numbers. However, specificity can be a problem for these assays with variation believed to come from the reverse transcription step. Specific priming has been compared between random hexamer and oligo(dT) primers, but the success rates appear to be largely template dependent. Primer-independent reverse transcription complicates strand-specific detection, resulting in large portions of the PCR product coming from the undesired strand. Writing in the April 2012 issue of BioTechniques, Feng et al. introduce a strand-specific RT-PCR methodology that relies on the use of reverse transcription primers where 4-7 adenine or thymine residues are replaced with cytosine or guanine, or vice versa, creating a 3-5oC change in melting temperature of the resulting PCR amplicon. Reverse transcription is then followed by gene specific qPCR and melting curve analysis. The secret to strand-specificity lies in those sequence modifications present on all primer-initiated cDNA transcripts since these allow quantitative separation of primer-initiated vs. non-specific primer-independent transcripts using melting curve analysis. Feng and his colleagues demonstrated this by quantifying the plus and minus strands of the PB2 gene during flu virus replication in cell culture. In the end, this new approach will prove valuable for researchers requiring accurate quantification of strand-specific mRNA expression.
Ana Henriques, Filipe Carvalho, Rita Pombinho, Olga Reis, Sandra Sousa, and Didier Cabanes, BioTechniques Rapid Dispatches, doi:10.2144/000113906
The ability of a pathogen to successfully infect a host depends on a number of variables. Identifying these critical virulence factors is of public health interest, but such efforts can be delayed by the need for expensive or complicated methods requiring highly trained personnel, dedicated facilities, or large numbers of animal models for testing. The search for virulence factors can be approached through the use of directed or random gene disruption strategies to generate mutant strains whose phenotypes can be analyzed in individual host animals. However, this approach requires many animals and sacrifices in terms of scale or specificity of the target genes. To provide an alternative that would allow the study of specifically targeted genes in a reduced number of animals, Henriques et al. designed a PCR-based method for virulence screening that provides a relative quantification of individual mutants from a pool of bacteria. The authors first cultured individual tagged bacterial mutants (insertion or deletion mutants) and then pooled their mutants in equal portions for inoculation into a group of mice. Following infection, the bacteria were recovered from mouse spleen and liver and the relative abundance of each mutant was calculated using conventional colony PCR with carefully designed primers. This new approach was able to easily distinguish virulence-attenuated mutants from the pool of recovered bacteria, demonstrating that PCR screening of pooled mutants is a rapid and sensitive means to initially identify virulence factors while significantly reducing the time and number of animals required.
It is clear to see from our 2012 PCR picks that PCR remains a versatile technique applicable to a wide range of studies that will likely continue to develop in the coming years as our research needs evolve.