Determining the sizes and measuring the quantities of DNA molecules are fundamental tasks in molecular biology. DNA sizes are usually evaluated by gel electrophoresis, but this method cannot simultaneously size and quantitate a DNA at low zeptomole (zmol) levels of concentration. We have recently developed a new technique, called bare-narrow-capillary/hydrodynamic-chromatography or BaNC-HDC, for resolving DNA based on their sizes without using any sieving matrices. In this report, we utilize BaNC-HDC for measuring the sizes and quantities of DNA fragments at zmol to several-molecule levels of concentration. DNA ranging from a few base pairs to dozens of kilo base pairs are accurately sized and quantitated at a throughput of 15 samples per hour, and each sample contains dozens of DNA strands of different lengths. BaNC-HDC can be a cost-effective means and an excellent tool for high-throughput DNA sizing and quantitation at extremely low quantity level.

Invited for the cover of this issue are the groups of Shaorong Liu at the University of Oklahoma and Meiping Zhao at Peking University. The top part of the image depicts a new but simple way to resolve DNA, which can be separated using only a piece of open narrow capillary; the bottom part depicts the high-throughput and high-resolution separation results. Read the full text of the article at 10.1002/chem.201403861.

Simultaneous determination of indole-3-acetic acid and methyl indole-3-acetic acid ester in small amounts of plant tissue is essential for elucidating their mutual transformation mechanism and the in vivo function of methyl indole-3-acetic acid ester. Rapid quantification of flavonoids in the same sample is important for clarifying their roles in the transport of auxins and other phytohormones. Herein, we describe a simple method for the simultaneous determination of indole-3-acetic acid and its methyl ester in the roots of the Arabidopsis thaliana seedlings and a protocol for the rapid extraction and quantification of quercetin and kaempferol in these seedlings. High-performance liquid chromatography coupled with electrospray ionization time-of-flight tandem mass spectrometry was used for the detection of all the compounds. Negative data for indole-3-acetic acid and positive data for methyl indole-3-acetic acid ester were collected in two successive files with a single injection of the extracted sample. Under optimized conditions, the limit of detection for the four compounds was 2 ng/mL for indole-3-acetic acid, 0.5 ng/mL for methyl indole-3-acetic acid ester, 5 ng/mL for quercetin, and 1 ng/mL for kaempferol, respectively. Because of the high sensitivity of the assay, only 2–10 mg of the plant material was required to obtain quantitative results.

DNA 3′-phosphatases play a unique role in the repair of strand breaks induced by DNA damaging agents, such as ionizing radiation or oxidative stress. In this paper, we present an efficient detection system for rapid screening of DNA 3′-phosphatases and their inhibitors. A unique template substrate has been designed to hybridize with the universal molecular beacon (U-MB), and the detection process is carried out in a quantitative real-time PCR. The method is successfully applied to monitor the activity and kinetics of two typical 3′-phosphatases, that is, T4 polynucleotide kinase phosphatase (PNKP) and calf intestinal alkaline phosphatase (CIP). The inhibition effect of heparin on T4 PNKP and theophylline on CIP is also quantitatively characterized. The proposed method is demonstrated to be very useful for sensitive, high-throughput, and precise measurement of various 3′-phosphatases and their inhibitors.

A novel molecularly imprinted hydrogel for bovine serum albumin (BSA) was prepared using cupric ion as the bridge between the template BSA and the functional monomer 4-vinylpyridine. N-Isopropylacrylamide (NIPA) was used as an assistant monomer to provide the stimuli-responsibility of the polymer. The adsorption conditions of BSA on the BSA-Cu(II)-imprinted hydrogel were optimized considering the influences of pH, temperature, and salt concentration. The proteins bound on the imprinted hydrogel can be easily recovered under mild conditions by using 10 mmol/L ethylene diamine tetraacetic acid (EDTA) (pH 7.0) containing 150 mmol/L NaCl as the eluting solution. The imprinting effect and adsorption capacity of the polymer were found to be significantly improved compared to the hydrogel prepared in the absence of cupric ion. The results demonstrated the advantages of using a template-metal ion-monomer coordination system to strengthen the interaction between the protein and monomer. The effects of different metals ions including Zn(II), Ni(II), Co(II), Cd(II), and Al(III) on the recognition ability of the BSA-Cu(II)-imprinted hydrogel were also investigated. The polymer showed high selectivity toward both the template protein and the cupric ion.

Stimuli-responsive protein imprinted polymers were obtained via a combination of molecular imprinting and reversible stimuli-responsive polymer using lysozyme or cytochrome c as template, N-isopropylacrylamide (NIPA) as major monomer, methacrylic acid (MAA) and acrylamide (AAm) as functional co-monomers, and N,N-methylenebisacrylamide (MBAAm) as crosslinker. The molecularly imprinted polymers (MIPs) can respond not only to external stimuli such as temperature and salt concentration, but also to the corresponding template protein with significant specific volume shrinking. This specific shrinking behavior was attributed to the synergistic effect of multiple-site weak interactions (electrostatic force, hydrogen bonding and hydrophobic interaction) and the cavity effect. The MIPs showed highly selective adsorption of template proteins with specific shrinking compared with the non-imprinted polymers. The results indicated that the MIPs seemed to change shape to accommodate the conformation of the template protein leading to the formation of a shape complementary cavity. Copyright © 2008 John Wiley & Sons, Ltd.