Polymerase Chain Reaction Isolation Research Articles

English

Rosemary plant is in high demand due to its application in traditional health care, food flavoring, fragrance and pharmaceutical industries. It contains high level of secondary metabolites which are responsible for its beneficial activities. Application of molecular techniques would facilitate the production of these substances and screening of accessions. The isolation of polymerase chain reaction (PCR) amplifiable genomic DNA is a pre-requisite for taking advantage of these technologies. Even though several DNA isolation protocols for plants with high level of secondary metabolites were developed, they may not permit optimal DNA extraction due to chemotypic variation within species. Extracting DNA from different rosemary accessions is a challenging task due to its high level of secondary metabolites. Therefore, this research is conducted with the aim of optimizing a reliable and rapid method suitable for extracting DNA from rosemary plants. The optimized protocol avoids the use of repeated toxic phenols, liquid nitrogen and large polypropylene tube. It is appropriate for both fresh and dry leaf samples. The quality of the obtained DNA was excellent as evident by A260/A280 ratio ranging from 1.7 to 1.89 and the concentration ranged from 195.8 to 2184 ng/µl. The success of this protocol indicated its applicability for other plants with high secondary metabolite contents.   Key words: DNA isolation, secondary metabolites, rosemary, gel electrophoresis, polymerase chain reaction (PCR) amplification.

Isolation of polymerase chain reaction-ready bacterial DNA from Lake Baikal sediments by carboxyl-functionalised magnetic polymer microspheres

Carboxyl group-containing magnetic nonporous poly(2-hydroxyethyl methacrylate- co-ethylene dimethacrylate) (P(HEMA- co-EDMA)) microspheres were used for the isolation of polymerase chain reaction (PCR)-ready DNA from samples of Baikal sediments. DNA was isolated using the phenol extraction method or the Soil DNA Isolation Kit. The occurrence of false-negative results in PCR caused by the presence of extracellular inhibitors in DNA samples was solved using solid phase reversible DNA immobilisation. PCR-ready DNA was reversibly adsorbed to the microspheres in the presence of 8.0% (w/v) poly(ethylene glycol) (PEG 6000) and 2.0 M sodium chloride concentrations. The adsorbed DNA was released from the microspheres in a low ionic strength TE buffer. The quality of isolated DNA was checked by PCR amplification.

Isolation and analysis of amplified cDNA fragments during detection of unknown polymorphisms with temperature modulated heteroduplex chromatography.

A procedure for isolation of polymerase chain reaction (PCR) fragments from an automated system for DNA fragment analysis is described. The technique is used for screening of unknown mutations or polymorphisms. Fragments are isolated during the analysis from the system by an automated unit for high-throughput projects or manually when working with a few samples.