PCR is a cornerstone of modern molecular biology. Even with its firm foothold in the lab, new PCR developments regularly appear in the scientific literature. This week, we take a look at the top ten BioTechniques PCR articles. Even if you are well-versed in PCR theory and application, take a look at our list and comments—you never know when you might find a new way to speed up that pesky experiment.
1. “Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids” by A. Bryksin and I. Matsumura
Bryksin and Matsumura’s article has remained one of the top downloads since its publication in 2010. The development of a simple methodology for generating recombinant plasmids with multiple pieces of DNA further illustrates the utility of PCR beyond simply amplifying sections of DNA for analysis or sequencing, PCR is a versitile tool that can also be used to join DNA fragments, create mutations and identify unknown genomic regions.
For additional information on PCR cloning, check out a commentary written by
Matsumura earlier this year on
the development of overlap extension techniques:
2. “Real-time PCR for mRNA quantification” by Wong and Medrano
Another frequently downloaded article, Wong and Medrano’s review of real-time PCR technologies for quantifying mRNA is a must-read for researchers using real-time PCR in their experiments.
Walsh et al.’s article was cited this year by the editors of BioTechniques as one of our 30th anniversary Gem selections for presenting a new and simple PCR methodology that expanded the technique's use even further.
miRNA continues to be of great interest to the research community. In March,
Redshaw et al. detailed their comparison of miRNA isolation techniques and
qPCR platforms to determine the effects of different approaches on
quantification accuracy and repeatability.
5. “Extraction of genomic DNA from yeasts for PCR-based applications” by Looke et al.
Methods for extracting DNA and RNA from various sources remain an important topic for researchers, especially those interested in high-throughput PCR applications. Here, Looke et al. developed a quick and effective methodology for extracting genomic DNA from yeast that is suitable for different PCR methodologies.
Since the description of inverse PCR, methods to isolate flanking sequences using PCR have continued to expand. This 2007 article by Liu and Chen describes an advance on the thermal asymmetric interlaced PCR (TAIL-PCR) method that generates larger sized products. The so called high efficiency TAIL-PCR (hiTAIL-PCR) generates major products in the size range of 1-3kb, enhancing researchers ability to identify flanking unknown regions.
7. “Direct PCR amplification and sequencing of specimens’ DNA from preservative ethanol” by Shokralla et al.
The ability to amplify target DNA from a limited amount of starting material is important for forensic and environmental PCR applications. Here, Shokralla et al. found that preservative ethanol from a stored specimen could be used as a starting material for PCR amplification. This enables scientists to analyze very precious samples without destroying the specimen.
8. “Method for isolation of PCR-ready genomic DNA from zebrafish tissue” by Meeker et al.
Zebrafish is an extremely important model organism in biology. This method and similar approaches are quick and simple techniques for confirming the presence of transgenes or genetic modifications when performing zebrafish studies.
In real-time PCR, understanding experimental variation and improving robustness is very important. In this 2005 article, The et al. describe an approach to reducing variation with double correction and second derivative calculations.
PCR inhibitors can be the thorn in a researchers side. In this article, King et al. tackle the issue of PCR inhibition as it relates to tricky ancient DNA extracts providing practical advice for ancient DNA specialists as well as others dealing with complex starting samples.