Thursday, July 29, 2010

Solutions for problematic plant templates

Recently, one of our customers (Millie Burrell of Texas A&M University) thanked us for helping her resurrect some DNA templates that she was on the “verge of abandoning due to lack of quality DNA and subsequent PCR amplification for downstream sequencing.” Through the use of the QuickExtract™ Seed Solution and PlantAmp™ PCR System, she was able to obtain PCR results for DNA extracted from Caulanthus amplexicaulus, a little-known wild relative of Arabidopsis. According to Millie, this plant is a “rare and endangered species that grows on serpentine (ultramafic) soils known for low macronutrient levels and conventionally toxic levels of heavy metals.” The QuickExtract Seed protocol uses up to 10 mg of ground/fragmented seed in an 8-minute protocol that is suitable for high-throughput workflows. The PlantAmp PCR System is formulated to optimize PCR amplification from samples containing polyphenols and other PCR inhibitors, in a convenient premix format.

Photo credit: joedecruyenaere

Wednesday, July 14, 2010

Fosmid cloning: Alive and kicking

Although advances in next-generation sequencing technology have replaced the need for clone libraries in many laboratories, fosmid libraries are still useful in a variety of functional genomics studies.

Xu et al.1 present the first report of a host-specific restriction system associated with S-modification of DNA (phosphorothioation), instead of methylation. The authors observed that the enteropathogenic Salmonella enterica serovar Cerro 87, which possesses S-modified DNA, restricts DNA isolated from E. coli, while protecting its own DNA by site-specific phosphorothioation. They located the gene in S. enterica by screening a genomic library created using the CopyControl™ Fosmid Library Production kit. The authors screened the fosmid libraries using degenerate gene cluster–specific primers to locate the genes responsible for phosphorothioation and also to perform mutational analysis. Their results establish a biological role for phosphorothioation activity, and suggest that DNA S-modification may act not only as protective system against bacteriophage infection, but also as an epigenetic signal for new biological functions.

In another study by Lucker et al.2, the CopyControl Kit was used to reconstruct the complete genome of Candidatus Nitrospira defluvii (Ca. N. defluvii) from a metagenomic fosmid library prepared from an activated sludge enrichment culture. The immense ecological and technical significance of Nitrospira contrasts with the scarce knowledge about these bacteria. Except for one 137-kb contig, genomic sequences from Nitrospira have not been obtained yet. This situation has been highly unsatisfactory because deeper insight into the biology of these elusive nitrate oxidizing bacteria is crucial for a better understanding of nitrogen cycling in natural and engineered systems. On the basis of this first-deciphered Nitrospira genome and experimental data, the authors show that Ca. N. defluvii differs fundamentally in its enzymatic repertoire and metabolic pathways from all other known nitrifying bacteria. The current study provides valuable insights into the evolution of nitrite oxidation.

ResearchBlogging.org1. Xu, T. et al. (2010). A novel host-specific restriction system associated with DNA backbone S-modification in Salmonella Nucleic Acids Research DOI: 10.1093/nar/gkq610
2. Lucker, S. et al. (2010). A Nitrospira metagenome illuminates the physiology and evolution of globally important nitrite-oxidizing bacteria Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1003860107

Friday, July 9, 2010

When is “direct PCR” not direct?

We’ve seen a few companies promoting “direct PCR” products, where a sample can be added directly to a PCR mixture without first extracting DNA. This seems like a good idea in theory, but the reality is not as simple--especially for samples that contain PCR inhibitors.

Therefore, companies that offer kits for direct PCR have had to lengthen these procedures to contend with inhibitors. For example, one kit requires NaOH treatment for 10 minutes, followed by neutralization with Tris-HCl. A large PCR volume is needed and an additive must be included in the gel loading buffer prior to electrophoresis. A second direct PCR kit also requires a large PCR volume and an additive in the loading buffer. In addition, since the polymerase is not Taq-based, annealing temperature must be carefully optimized. For this kit, alternate protocols are offered for challenging templates or multiplex PCR consisting of adding Dilution Buffer and Additive, followed by vortexing, centrifugation, two incubations, and a second centrifugation.

Both kit protocols described above are based on processing animal tissue samples. For plant tissue, even more steps are needed to remove inhibitors.

In contrast, Epicentre’s QuickExtract™ products offer a rapid, efficient method to extract nucleic acids from virtually any sample for PCR-based assays, with most samples being processed in only 8 minutes. Our guaranteed FailSafe™ PCR System ensures reliable PCR results, the first time and every time. Together, these products provide a simpler and more effective method than direct PCR.

Special offer: Order the FailSafe PCR PreMix Selection Kit and get 5 ml of any QuickExtract product free.

Tuesday, July 6, 2010

Gene expression analysis from Ribo-Zero™-treated RNA

The Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat) was compared to a competitive kit's performance in removing rRNA from total RNA samples that were used for RNA-Seq. Both kits performed equivalently for intact RNA samples, while the Ribo-Zero Kit performed better than the competitive kit for partially degraded RNA samples.

A

B

C

Consistent gene expression data from Ribo-Zero™-treated RNA. Intact and partially degraded Universal Human Reference RNA (UHRR) was treated with either the Ribo-Zero Kit or a competitive kit. The rRNA-depleted RNAs were then used to prepare cDNA libraries that were sequenced on an Illumina® GAII sequencer.  A) Number of genes detected in libraries prepared from intact or partially degraded, rRNA-depleted UHRR. B) Correlation of expression levels for intact RNA. C) Correlation of expression levels for partially fragmented RNA. RPKM, reads per kilobase of exon model per million mapped reads.

(Click images to enlarge them.)