Recycling Isolation of Plant DNA,A Novel Method

DNA is one of the most basic and essential genetic materials in the field of molecular biology.To date,isolation of sufficient and good-quality DNA is still a challenge for many plant species,though various DNA extraction methods have been published.In the present paper,a recycling DNA extraction method was proposed.The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times,and correspondingly four DNA precipitations(termed as the 1st,2nd,3rd and 4th DNA sample, respectively) were conducted.This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method.This modified CTAB method was tested in eight plant species,wheat,sorghum,barley,corn,rice,Brachypodium distachyon,Miscanthus sinensis and tung tree.The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species.The DNA samples were good templates for PCR amplification of both ISSR and SSR markers.The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods,and thus is an effective and universal DNA isolation method.     

iCatch:a new strategy for capturing large DNA fragments using homing endonucleases

Natural genetic materials contain many biosynthetic gene clusters encoding potentially valuable natural products,many of which can be used directly without codon optimization or other manipulations.With the development of synthetic biology,several DNA assembly standards have been proposed,conveniently facilitating the reuse of natural materials.Among these standards,the iBrick assembly standard was developed by our laboratory to manipulate large DNA fragments,employing two homing endonucleases.Considering the difficulty of cloning large iBrick parts using conventional endonuclease-mediated restriction and ligation methods,we herein present a new method,known as iCatch,which readily captures biosynthetic gene clusters.As the clusters cloned by iCatch have the prefix and suffix of the iBrick standard,they serve as new iBrick parts and are therefore conducive to further editing and assembly with the iBrick standard.iCatch employs the natural homologous recombination system to flank the region of interest with I-Scel and PI-Pspl recognition sites,after which the genome is digested with I-Scel or PI-Pspl and the fragments are then self-ligated to clone the target DNA fragments.We used this method to successfully capture the actinorhodin biosynthetic cluster from Streptomyces coelicolor and then heterologously expressed this cluster in a thermophilic Streptomyces strain.We propose that iCatch can be used for the cloning of DNA sequences that are dozens of kilobases in length,facilitating the heterologous expression of microbial natural products.Moreover,this cloning methodology can be a complementary tool for the iBrick standard,especially in applications requiring the manipulation of large DNA fragments.     

依赖粪便材料的大熊猫肠道耶尔森氏菌的检测

It is inevitable to develop noninvasive sampling methods to do studies on giant panda even diagnose the diseases since which is so endangered that it's impossible to carry out invasive sampling. A non-invasive sampling method to detect the intestinal pathogen, Yersirda enterocolitica in feces of pandas based designing PCR primers was established in this study. The main procedures are based on bacteria enrichment and cell lysis before binding the pathogen DNA to silica powder at high concentration of Kalium iodide and neutral pH conditions. Before PCR cycles, the binded DNA is washed with 80% ethanol and eluted with diluted EDTA buffer. Restdts showed that the silica-based feces DNA-purification method could remove the inhibitors of PCR so applicable to detect the target pathogen.     

A hot start alternative for high-fidelity DNA polymerase amplification mediated by quantum dots

Quantum dots （QDs） are of great interest due to their unique chemical and physical properties. Recently, a hot start （HS） polymerase chain reaction （PCR） amplification performance based on QDs with a high-fidelity Pfu DNA polymerase has been reported. However, whether QDs can trigger HS effects with other high-fidelity or conventional DNA polymerases is yet to be understood. In the present study, we studied the QD-triggered HS effects with four high-fidelity and three conventional DNA polymerases, and the HS effect comparisons among them were also made. It was found that QDs could trigger a distinct HS PCR amplification performance with all the four tested high,fidelity DNA polymerases, and specific target DNA could be well amplified even if the PCR mixture was preincubated for 2 h at 50℃. On the contrary, the HS effects were not prominent with all the three conventional Taq DNA polymerases. Specifically, the fidelity of Pfu is not sacrificed in the presence of QDs, even after a 1 h pre-incu- bation at 50℃ before PCR. Furthermore, the electrophoresis results preliminarily demonstrated that QDs prefer to adsorb high-fidelity polymerases rather than conventional ones, which might result in the QD-triggered HS effects on PCR performance by using high-fidelity DNA poly- merases.     

Special Issue on “Gene Regulatory Network”

The journal Genomics Proteomics & Bioinformatics (GPB) is now inviting submissions for a special issue (to be published around Dec 2012) on the topic of "Gene Regulatory Network". The Gene Regulatory Network (GRN) is a collection of DNA sequences which interact with each other and other cellular components, thereby governing the rates at which genes in the network are transcribed to change the genomic activity. GRNs are evolutionally-conserved and play critical roles in various biological processes and attract more and more research interest.     

An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction

Microbes are ubiquitously distributed in nature, and recent culture-independent studies have highlighted the significance of gut microbiota in human health and disease. Fecal DNA is the primary source for the majority of human gut microbiome studies. However, further improvement is needed to obtain fecal metagenomic DNA with sufficient amount and good quality but low host genomic DNA contamination. In the current study, we demonstrate a quick, robust, unbiased,and cost-effective method for the isolation of high molecular weight(23 kb) metagenomic DNA(260/280 ratio 1.8) with a good yield(55.8 ± 3.8 ng/mg of feces). We also confirm that there is very low human genomic DNA contamination(eubacterial: human genomic DNA marker genes = 2~(27.9):1) in the human feces. The newly-developed method robustly performs for fresh as well as stored fecal samples as demonstrated by 16 S r RNA gene sequencing using 454 FLX+.Moreover, 16 S r RNA gene analysis indicated that compared to other DNA extraction methods tested, the fecal metagenomic DNA isolated with current methodology retains species richnessand does not show microbial diversity biases, which is further confirmed by q PCR with a known quantity of spike-in genomes. Overall, our data highlight a protocol with a balance between quality,amount, user-friendliness, and cost effectiveness for its suitability toward usage for cultureindependent analysis of the human gut microbiome, which provides a robust solution to overcome key issues associated with fecal metagenomic DNA isolation in human gut microbiome studies.     

Micro Flow-Through Thermocycler with Simple Meandering Channel with Symmetric Temperature Zones for Disposable PCR-Devices in Microscope Slide Format

Chip-based flow-through PCR implements the PCR as a continuous process for nucleic acid analytics. The sample is transported in a winding channel through temperature zones required for denaturation, annealing and extension. Main fields of application are the monitoring of continuous processes for rapid identification of contaminants and quality control as well as high throughput screening of cells or microorganisms. A modular arrangement with five heating zones for flow-through PCR is discussed and evaluated. The special heater arrangement allows the implementation of up to 40 cycles on the footprint of a microscope slide, which is placed on top ofa 5 zones heating plate. Liquid/liquid two phase flow of PCR reaction mixture and mineral oil have been applied to create a segmented flow process scheme. In that way, the developed system may provide flow-through PCR as a unit operation for the droplet based microfluidics platform. The single use of disposable devices is commonly preferred due to the sensitivity of the PCR process to contaminations. All-glass microfluidic chips and disposable chip devices, made from polycarbonate as a replication with identically geometry, have been fabricated and tested. For the first time, microchannel geometries with nearly circular profile developed by all-glass technology have been transferred to mass fabrication by injection compression molding. Both devices have been successfully applied for the detection of the tumor suppressor gene p53. Although product yield and selectivity of the amplification process do not depend on the chip material, a well defined, reliable segmented flow regime could only be realized in the all-glass chip.     

Using MutPred derived mtDNA load scores to evaluate mtDNA variation in hypertension and diabetes in a two-population cohort:The SABPA study

Mitochondrial DNA(mt DNA) variation has been implicated in many common complex diseases, but inconsistent and contradicting results are common. Here we introduce a novel mutational load hypothesis, which also considers the collective effect of mainly rare variants, utilising the Mut Pred Program.We apply this new methodology to investigate the possible role of mt DNA in two cardiovascular disease(CVD) phenotypes(hypertension and hyperglycaemia), within a two-population cohort(n = 363; mean age 45 ± 9 yrs). Very few studies have looked at African mt DNA variation in the context of complex disease, and none using complete sequence data in a well-phenotyped cohort. As such, our study will also extend our knowledge of African mt DNA variation, with complete sequences of Southern Africans being especially under-represented. The cohort showed prevalence rates for hypertension(58.6%) and prediabetes(44.8%). We could not identify a statistically significant role for mt DNA variation in association with hypertension or hyperglycaemia in our cohort. However, we are of the opinion that the method described will find wide application in the field, being especially useful for cohorts from multiple locations or with a variety of mt DNA lineages, where the traditional haplogroup association method has been particularly likely to generate spurious results in the context of association with common complex disease.